Anti-CD40 antibodies and uses thereof

ABSTRACT

An isolated monoclonal antibody that specifically binds human CD40. A nucleic acid molecule encoding the antibody, an expression vector, a host cell and a method for expressing the antibody are also provided. The present invention further provides an immunoconjugate, a bispecific molecule, a chimeric antigen receptor, an oncolytic virus and a pharmaceutical composition comprising the antibody, as well as a treatment method using an anti-CD40 antibody of the invention.

FIELD OF THE INVENTION

The invention relates to an antibody specifically binding to human CD40,preparation and use thereof, especially its use in treatment of humandiseases associated with CD40, such as cancers, inflammatory diseases,infectious diseases, atherothrombosis, and respiratory diseases.

BACKGROUND OF THE INVENTION

CD40, also referred to as tumor necrosis factor receptor superfamilymember 5 or TNFR5, is a transmembrane costimulatory protein expressed onantigen presenting cells such as B cells, macrophages, and dendriticcells. Binding of this protein with CD40L (CD154), the major ligandexpressed primarily by activated T lymphocytes and platelets, activatesantigen presenting cells and triggers a variety of downstreamsignalings, including immune cell activation and proliferation, andproduction of cytokines and chemokines, enhancing cellular and immunefunctions (Ara A et al., (2018) Immunotargets Ther 7: 55-61).

On the other hand, CD40 is also found on non-immune cells and tumors(Costello et al., (1999) Immunol Today 20(11): 488-493; Tong et al.,(2003) Cancer Gene Ther 10(1): 1-13; Lee et al., (2014) Curr Cancer DrugTargets 14(7): 610-620; Ara A et al., (2018) supra), and was reported tobe involved in pathologies of several inflammatory diseases, includingautoimmune diseases, atherothrombosis, cancers, and respiratorydiseases. For example, CD40/CD40L expression was up-regulated inatheroma-associated cells. CD40 was found in neally all B-cellmalignancies and up to 70% of solid tumors, and CD40 engagement incertain B-cell malignancies caused increased expression of many factorsthat protect the cell from apoptosis induced by apoptotic agents (Lee etal., (1999) Proc Natl Acad Sci USA 96:9136-9141).

Despite of CD40's complicated effects on tumor development, severalanti-CD40 antibodies have been developed for potential tumor treatment.CP-870,893, a fully human IgG2 CD40 agonistic antibody developed byPfizer, can activate dendritic cells and has shown clinical efficacy ina number of settings of patients with advanced cancers (Vonderheide etal., (2007) J Clin Oncol 25(7): 876-883; Gladue et al., (2011) CancerImmunol Immunother 60(7): 1009-1017; Beatty et al., (2013) Expert RevAnticancer Ther 17(2): 175-186; Vonderheide et al., (2013)Oncoimmunology 2(1): e23033; Nowak et al., Ann Oncol 26(12): 2483-2490;2015 U.S. Pat. No. 7,338,660). Dacetuzumab, also known as SGN-40, ahumanized lgG1 agonistic anti-CD40 antibody developed by SeattleGenetics, has also shown anti-tumor activity when given intravenouslyevery week, especially in patients with diffuse large B-cell lymphoma.Preclinical data also showed synergic effect of Dacetuzumab with otheragents such as the anti-CD20 mAb rituximab (Lapalombella et al., (2009)Br J Haematol 144(6): 848-855; Hussein et al., (2010) Haematologica95(5): 845-848; de Vos et al., (2014) J Hematol Oncol 7: 44). Chi Lob7/4, another chimeric anti-human IgG1 agonistic anti-CD40 antibodydeveloped by Cancer Research UK, is undergoing initial clinical testing.Eleven of the 21 patients showed stable disease with no complete orpartial responses (Chowdhury et al., (2014) Cancer Immunol Res 2(3):229-240). Further, antagonistic anti-CD40 antibodies have been studiedfor their anti-tumor activity on human multiple myeloma and chroniclymphocytic leukemia (Bensinger W et al., (2012) Br J Haematol. 159(1):58-66; Mohammad Luqman et al., (2008) Blood 112: 711-720).

There remains a need for more CD40 antibodies with improvedpharmaceutical characteristics.

SUMMARY OF THE INVENTION

The present invention provides an isolated monoclonal antibody, forexample, a mouse, human, chimeric or humanized monoclonal antibody, thatbinds to CD40 (e.g., the human CD40, and monkey CD40). It may be anagonistic CD40 antibody that activates CD40 signaling.

The antibody of the invention can be used for a variety of applications,including detection of the CD40 protein, and treatment and prevention ofCD40 associated diseases, such as cancers, inflammatory diseases,infectious diseases, atherothrombosis, and respiratory diseases.

Accordingly, in one aspect, the invention pertains to an isolatedmonoclonal antibody (e.g., a humanized antibody), or an antigen-bindingportion thereof, that binds CD40, having a heavy chain variable regionthat comprises a CDR1 region, a CDR2 region and a CDR3 region, whereinthe CDR1 region, the CDR2 region and the CDR3 region comprise amino acidsequences having at least 80%, 85%, 90%, 95%, 98%, or 99% identity to,or set forth in (1) SEQ ID NOs: 1, 8 and 15, respectively; (2) SEQ IDNOs: 1, 9 and 15, respectively; (3) SEQ ID NOs: 2, 9 and 15,respectively; (4) SEQ ID NOs: 3, 10 and 16, respectively; (5) SEQ IDNOs: 4, 11 and 17, respectively; (6) SEQ ID NOs: 5, 12 and 18,respectively; (7) SEQ ID NOs: 6, 13 and 19, respectively; or (8) SEQ IDNOs: 7, 14 and 20, respectively; wherein, the antibody, orantigen-binding fragment thereof, binds to CD40.

In one aspect, an isolated monoclonal antibody, or an antigen-bindingportion thereof, of the present invention comprises a heavy chainvariable region comprising an amino acid sequence having at least 80%,85%, 90%, 95%, 98% or 99% identity to, or set forth in SEQ ID NOs: 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50, wherein theantibody or antigen-binding fragment thereof binds to CD40.

In one aspect, an isolated monoclonal antibody, or an antigen-bindingportion thereof, of the present invention comprises a light chainvariable region that comprises a CDR1 region, a CDR2 region and a CDR3region, wherein the CDR1 region, the CDR2 region, and the CDR3 regioncomprise amino acid sequences having at least 80%, 85%, 90%, 95%, 98% or99% identity to, or set forth in (1) SEQ ID NOs: 21, 27 and 31,respectively; (2) SEQ ID NOs: 22, 28 and 32, respectively; (3) SEQ IDNOs: 23, 29 and 33, respectively; (4) SEQ ID NOs: 24, 27 and 34,respectively; (5) SEQ ID NOs: 25, 27 and 35, respectively; or (6) SEQ IDNOs: 26, 30 and 36, respectively; wherein the antibody orantigen-binding fragment thereof binds to CD40.

In one aspect, an isolated monoclonal antibody, or an antigen-bindingportion thereof, of the present invention comprises a light chainvariable region comprising an amino acid sequence having at least 80%,85%, 90%, 95%, 98% or 99% identity to, or set forth in SEQ ID NOs: 51,52, 53, 54, 55, 56, 57, 58, 59, 60, or 61, wherein the antibody orantigen-binding fragment thereof binds to CD40.

In one aspect, an isolated monoclonal antibody, or an antigen-bindingportion thereof, of the present invention comprises a heavy chainvariable region and a light chain variable region each comprises a CDR1region, a CDR2 region and a CDR3 region, wherein the heavy chainvariable region CDR1, CDR2 and CDR3, and the light chain variable regionCDR1, CDR2 and CDR3 comprise amino acid sequences having at least 80%,85%, 90%, 95%, 98% or 99% identity to, or set forth in (1) SEQ ID NOs:1, 8, 15, 21, 27 and 31, respectively; (2) SEQ ID NOs: 1, 9, 15, 21, 27and 31, respectively; (3) 2, 9, 15, 21, 27 and 31, respectively; (4) SEQID NOs: 3, 10, 16, 22, 28 and 32, respectively; (5) SEQ ID NOs: 4, 11,17, 23, 29 and 33, respectively; (6) SEQ ID NOs: 5, 12, 18, 24, 27 and34, respectively; (7) SEQ ID NOs: 6, 13, 19, 25, 27 and 35,respectively; or (8) SEQ ID NOs: 7, 14, 20 26, 30 and 36, respectively,wherein the antibody or antigen-binding fragment thereof binds to CD40.

In one embodiment, an isolated monoclonal antibody, or theantigen-binding portion thereof, of the present invention comprises aheavy chain variable region and a light chain variable region, the heavychain variable region and the light chain variable region comprisingamino acid sequences having at least 80%, 85%, 90%, 95%, 98% or 99%identity to, or set forth in (1) SEQ ID NOs: 37 and 51, respectively;(2) SEQ ID NOs: 38 and 52, respectively; (3) SEQ ID NOs: 39 and 53,respectively; (4) SEQ ID NOs: 40 and 54, respectively; (5) SEQ ID NOs:41 and 55, respectively; (6) SEQ ID NOs: 44 and 58, respectively; (7)SEQ ID NOs: 45 and 59, respectively; (8) SEQ ID NOs: 46 and 60,respectively; (9) SEQ ID NOs: 46 and 61, respectively; (10) SEQ ID NOs:47 and 60, respectively; (11) SEQ ID NOs: 47 and 61, respectively; (12)SEQ ID NOs: 48 and 59, respectively; (13) SEQ ID NOs: 49 and 60,respectively; (14) SEQ ID NOs: 49 and 61, respectively; (15) SEQ ID NOs:50 and 60, respectively; (16) SEQ ID NOs: 50 and 61, respectively; (17)SEQ ID NOs: 42 and 56, respectively; or (18) SEQ ID NOs: 43 and 57,respectively, wherein the antibody or antigen-binding fragment thereofbinds to CD40.

In one embodiment, an isolated monoclonal antibody, or theantigen-binding portion thereof, of the present invention comprises aheavy chain and a light chain, the heavy chain comprising a heavy chainvariable region and a heavy chain constant region, the light chaincomprising a light chain variable region and a light chain constantregion, wherein, the heavy chain constant region comprises amino acidsequences having at least 80%, 85%, 90%, 95%, 98% or 99% identity to, orset forth in SEQ ID Nos: 62, 63 or 64, and the light chain constantregion comprises amino acid sequences having at least 80%, 85%, 90%,95%, 98% or 99% identity to, or set forth in SEQ ID Nos: 65 or 66, andthe heavy chain variable region and the light chain variable regioncomprise amino acid sequences described above, wherein the antibody orantigen-binding fragment thereof binds to CD40.

The antibody of the present invention in some embodiments comprises orconsists of two heavy chains and two light chains, wherein each heavychain comprises the heavy chain constant region, heavy chain variableregion or CDR sequences mentioned above, and each light chain comprisesthe light chain constant region, light chain variable region or CDRsequences mentioned above, wherein the antibody binds to CD40. Theantibody of the invention can be a full-length antibody, for example, ofan IgG1, IgG2 or IgG4 isotype. The antibody of the present invention inother embodiments may be a single chain antibody, or consists ofantibody fragments, such as Fab or Fab′2 fragments.

The antibody, or antigen-binding fragment, of the present inventionbinds specifically to human and monkey CD40, and blocks or promotesCD40-CD40L interaction. Agonistic CD40 antibodies of the presentinvention, able to activate CD40 signaling and drive maturation ofimmune cells such as dendritic cells, have in vivo anti-tumor effectcomparable to or better than prior art anti-CD40 agnostic antibodies,with equal or less toxicity. Tumors would not grow, or even totallyvanish, even after antibody administration has stopped.

The invention also provides an immunoconjugate comprising an antibody ofthe invention, or antigen-binding portion thereof, linked to atherapeutic agent, such as a cytotoxin. The invention also provides abispecific molecule comprising an antibody, or antigen-binding portionthereof, of the invention, linked to a second functional moiety (e.g., asecond antibody) having a different binding specificity than saidantibody, or antigen-binding portion thereof. In another aspect, theantibody or an antigen binding portions thereof of the present inventioncan be made into part of a chimeric antigen receptor (CAR). The antibodyor an antigen binding portions thereof of the present invention can alsobe encoded by or used in conjuction with an oncolytic virus.

Compositions comprising an antibody, or antigen-binding portion thereof,or immunoconjugate, bispecific molecule, or CAR of the invention, and apharmaceutically acceptable carrier, are also provided.

Nucleic acid molecules encoding the antibodies, or antigen-bindingportions thereof, of the invention are also encompassed by theinvention, as well as expression vectors comprising such nucleic acidsand host cells comprising such expression vectors. A method forpreparing an anti-CD40 antibody using the host cell comprising theexpression vector is also provided, comprising steps of (i) expressingthe antibody in the host cell and (ii) isolating the antibody from thehost cell or its cell culture.

In another aspect, the invention provides a method for enhancing animmune response in a subject, comprising administering to the subject atherapeutically effective amount of the antibody, or antigen-bindingportion thereof, of the invention. In some embodiments, the methodcomprises administering a composition, a bispecific molecule, animmunnoconjugate, a CAR-T cell, or an antibody-encoding orantibody-bearing oncolytic virus of the invention.

In another aspect, the invention provides a method for treatinginflammatory diseases, infectious diseases, atherothrombosis, orrespiratory diseases in a subject in need thereof, comprisingadministering to the subject a therapeutically effective amount of theantibody, or antigen-binding portion thereof, of the invention. In someembodiments, the method comprises administering a composition, abispecific molecule, an immunnoconjugate, a CAR-T cell, or anantibody-encoding or antibody-bearing oncolytic virus of the invention.In some embodiments, additional agents can be administered with theantibody, or an antigen-binding portion thereof, of the invention, suchas anti-inflammatory agents and antimicrobial agents. In someembodiments, the inflammatory diseases include autoimmune diseases.

In yet another aspect, the invention provides a method for preventing,treating or ameliorating a cancer disease in a subject, comprisingadministering to the subject a therapeutically effective amount of theantibody, or antigen-binding portion thereof, of the invention. Thecancer may be a solid or non-solid tumor, including, but not limited to,B cell lymphoma, chronic lymphocytic leukemia, multiple myeloma,melanoma, colon adenocarcinoma, pancreas cancer, colon cancer, gastricintestine cancer, prostate cancer, bladder cancer, kidney cancer, ovarycancer, cervix cancer, breast cancer, lung cancer, and nasopharynxcancer. In some embodiments, the method comprises administering acomposition, a bispecific molecule, an immunnoconjugate, a CAR-T cell,or an antibody-encoding or antibody-bearing oncolytic virus of theinvention. In some embodiments, at least one additional anti-cancerantibody can be administered with the antibody, or an antigen-bindingportion thereof, of the invention, such as an anti-VISTA antibody(antibody against the protein V-domain immunoglobulin (Ig) suppressor ofT-cell activation (VISTA; programmed death 1 homolog; PD1H; PD-1H)), ananti-PD-1 antibody, an anti-PD-L1 antibody, an anti-LAG-3 antibodyand/or an anti-CTLA-4 antibody. In yet another embodiment, an antibody,or an antigen-binding portion thereof, of the invention is administeredwith a cytokine (e.g., IL-2 and/or IL-21), or a costimulatory antibody(e.g., an anti-CD137 and/or anti-GITR antibody). In another embodiment,an antibody, or an antigen-binding portion thereof, of the invention isadministered with a chemotherapeutic agent, which may be a cytotoxicagent, such as epirubicin, oxaliplatin, and/or 5-fluorouracil (5-FU).The antibodies of the present invention can be, for example, mouse,human, chimeric or humanized antibodies.

Other features and advantages of the instant disclosure will be apparentfrom the following detailed description and examples, which should notbe construed as limiting. The contents of all references, Genbankentries, patents and published patent applications cited throughout thisapplication are expressly incorporated herein by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the agonistic activity ranking of 108 hybridoma clones.

FIGS. 2A and 2B show the promotional or inhibitory effect of anti-CD40antibodies on CD40/CD40L interaction, wherein antibodies 16A, 7B4 and13A promoted CD40/CD40L interaction (A) while antibody 92F6 inhibitedCD40/CD40L interaction (B).

FIG. 3 shows the agonistic activity of anti-CD40 antibodies.

FIG. 4A-4C show the anti-CD40 antibodies' involvement in dendritic cellmaturation as measured by staining of CD86 (A), CD80 (B) and CD83 (C).

FIGS. 5A and 5B show the binding capacity of the chimeric anti-CD40antibodies to human CD 40 (A) or monkey CD40 (B) expressed on HEK293Acells.

FIG. 6 shows the agonistic activity of the chimeric anti-CD40antibodies.

FIG. 7 shows the anti-CD40 antibodies' involvement in dendritic cellmaturation as measured by staining of CD86.

FIG. 8A-8D show the binding capacity of chimeric and humanized anti-CD40antibodies to human, or monkey CD40, wherein chimeric and humanrized13A2 antibodies (A) and humanized 7B4 antibodies (B) bound to humanCD40, and chimeric and humanrized 13A2 antibodies (C) and humanized 7B4antibodies (D) bound to monkey CD40.

FIGS. 9A and 9B show the agonistic activity of chimeric and humanrized7B4 antibodies (A) and chimeric and humanrized 13A2 antibodies (B).

FIG. 10A-10C show the anti-CD40 antibodies' involvement in maturation ofdendritic cells from donor 1 as measured by staining of CD86 (A), CD80(B) and CD83 (C).

FIGS. 11A and 11B show the anti-CD40 antibodies' involvement inmaturation of dendritic cells from donor 2 as measured by staining ofCD86 (A) and CD80 (B).

FIG. 12A-12C show the anti-CD40 antibodies' involvement in maturation ofdendritic cells from donor 3 as measured by staining of CD86 (A), CD80(B) and IL-12 (C).

FIG. 13A-13N show the binding affinity of chimeric and humanizedanti-CD40 antibodies 7B4 (A), 7B4-VH0VL0 (B), 7B4-VH2VL2 (C), 7B4-VH2VL3(D), 7B4-VH3VL2 (E), 7B4-VH3VL3 (F), 13A2 (G), 13A2-VH0VL0 (H),13A2-VH2VL2 (I), 13A2-VH2VL3 (J), 13A2-VH3VL2 (K) and 13A2-VH3VL3 (L) aswell as reference antibodies RO7009789 (M) and ADC1013 (N) to human CD40as measured by SPR.

FIGS. 14A and 14B show the binding capacity of chimeric and humanizedanti-CD40 antibodies to full-length CD40-ECD or its truncants (A) and tofull-length CD40-ECD or its mutants (B).

FIGS. 15A and 15B show the binding specificity of humanized anti-CD40antibodies 7B4-VH2VL2 (A) and 13A2-VH3VL3 (B) to human CD40.

FIGS. 16A, 16B and 16C show the engineered anti-CD40 antibodies'involvement in dendritic cell maturation as measured by staining of CD86(A), CD80 (B) and CD83(C).

FIG. 17A-17F show the average tumor volume in each group (A) andindividual tumor volumes in group administered with vehicle (B),7B4VH2VL2 (C), 3A2VH3VL3 (D), RO7009789 (E) or APX005 (F).

FIG. 18 shows the average animal body weight in group administered withhumanized anti-CD40 antibodies of the invention or control agents.

FIGS. 19A and 19B show the in vivo effect of humanized anti-CD40antibodies on tumor infiltrating CD45+CD3+CD4+ T cell (A) andCD45+CD3+CD8+ T cell (B) proliferation.

FIG. 20 shows the in vivo effect of humanized anti-CD40 antibodies ontumor infiltrating dentritic cell (CD45 positive and CD11c positivecell) maturation as measured by staining of CD86, CD80 and CD83.

DETAILED DESCRIPTION OF THE INVENTION

To ensure that the present disclosure may be more readily understood,certain terms are first defined. Additional definitions are set forththroughout the detailed description.

The term “CD40” refers to tumor necrosis factor receptor superfamilymember 5. The term “CD40” comprises variants, isoforms, homologs,orthologs and paralogs. For example, an antibody specific for a humanCD40 protein may, in certain cases, cross-react with a CD40 protein froma species other than human, such as monkey. In other embodiments, anantibody specific for a human CD40 protein may be completely specificfor the human CD40 protein and exhibit no cross-reactivity to otherspecies or of other types, or may cross-react with CD40 from certainother species but not all other species.

The term “human CD40” refers to an CD40 protein having an amino acidsequence from a human, such as the amino acid sequence of human CD40having a Genbank accession number of NP_001241.1 (SEQ ID NO.:68). Theterms “monkey or rhesus CD40” and “mouse CD40” refer to monkey and mouseCD40 sequences, respectively, e.g. those with the amino acid sequenceshaving Genbank Accession Nos. NP_001252791.1 (SEQ ID NO.:70) andNP_035741.2 (SEQ ID NO.:72), respectively.

The term “antibody” as referred to herein includes whole antibodies andany antigen binding fragment (i.e., “antigen-binding portion”) or singlechains thereof. Whole antibodies are glycoproteins comprising at leasttwo heavy (H) chains and two light (L) chains inter-connected bydisulfide bonds. Each heavy chain is comprised of a heavy chain variableregion (abbreviated herein as V_(H)) and a heavy chain constant region.The heavy chain constant region is comprised of three domains, C_(H1),C_(H2) and C_(H3). Each light chain is comprised of a light chainvariable region (abbreviated herein as VL) and a light chain constantregion. The light chain constant region is comprised of one domain, CL.The V_(H) and V_(L) regions can be further subdivided into regions ofhypervariability, termed complementarity determining regions (CDR),interspersed with regions that are more conserved, termed frameworkregions (FR). Each V_(H) and V_(L) is composed of three CDRs and fourFRs, arranged from amino-terminus to carboxy-terminus in the followingorder: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of theheavy and light chains contain a binding domain that interacts with anantigen. The constant regions of the antibodies can mediate the bindingof the immunoglobulin to host tissues or factors, including variouscells of the immune system (e.g., effector cells) and the firstcomponent (C1q) of the classical complement system.

The term “antigen-binding portion” of an antibody (or simply “antibodyportion”), as used herein, refers to one or more fragments of anantibody that retain the ability to specifically bind to an antigen(e.g., a CD40 protein). It has been shown that the antigen-bindingfunction of an antibody can be performed by fragments of a full-lengthantibody. Examples of binding fragments encompassed within the term“antigen-binding portion” of an antibody include (i) a Fab fragment, amonovalent fragment consisting of the V_(L) V_(H), C_(L) and C_(H1)domains; (ii) a F(ab′)₂ fragment, a bivalent fragment comprising two Fabfragments linked by a disulfide bridge at the hinge region; (iii) a Fdfragment consisting of the V_(H) and C_(H1) domains; (iv) a Fv fragmentconsisting of the V_(L) and V_(H) domains of a single arm of anantibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546),which consists of a V_(H) domain; (vi) an isolated complementaritydetermining region (CDR); and (viii) a nanobody, a heavy chain variableregion containing a single variable domain and two constant domains.Furthermore, although the two domains of the Fv fragment, V_(L) andV_(H), are coded by separate genes, they can be joined, usingrecombinant methods, by a synthetic linker that enables them to be madeas a single protein chain in which the V_(L) and V_(H) regions pair toform monovalent molecules (known as single chain Fv (scFv); see e.g.,Bird et al., (1988) Science 242:423-426; and Huston et al., (1988) Proc.Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodies arealso intended to be encompassed within the term “antigen-bindingportion” of an antibody. These antibody fragments are obtained usingconventional techniques known to those with skill in the art, and thefragments are screened for utility in the same manner as are intactantibodies.

An “isolated antibody”, as used herein, is intended to refer to anantibody that is substantially free of other antibodies having differentantigenic specificities (e.g., an isolated antibody that specificallybinds a CD40 protein is substantially free of antibodies thatspecifically bind antigens other than CD40 proteins). An isolatedantibody that specifically binds a human CD40 protein may, however, havecross-reactivity to other antigens, such as CD40 proteins from otherspecies. Moreover, an isolated antibody can be substantially free ofother cellular material and/or chemicals.

The terms “monoclonal antibody” or “monoclonal antibody composition” asused herein refer to a preparation of antibody molecules of singlemolecular composition. A monoclonal antibody composition displays asingle binding specificity and affinity for a particular epitope.

The term “mouse antibody”, as used herein, is intended to includeantibodies having variable regions in which both the framework and CDRregions are derived from mouse germline immunoglobulin sequences.Furthermore, if the antibody contains a constant region, the constantregion also is derived from mouse germline immunoglobulin sequences. Themouse antibodies of the invention can include amino acid residues notencoded by mouse germline immunoglobulin sequences (e.g., mutationsintroduced by random or site-specific mutagenesis in vitro or by somaticmutation in vivo). However, the term “mouse antibody”, as used herein,is not intended to include antibodies in which CDR sequences derivedfrom the germline of another mammalian species have been grafted ontomouse framework sequences.

The term “chimeric antibody” refers to an antibody made by combininggenetic material from a nonhuman source with genetic material from ahuman being. Or more generally, a chimetic antibody is an antibodyhaving genetic material from a certain species with genetic materialfrom another species.

The term “humanized antibody”, as used herein, refers to an antibodyfrom non-human species whose protein sequences have been modified toincrease similarity to antibody variants produced naturally in humans.

The phrases “an antibody recognizing an antigen” and “an antibodyspecific for an antigen” are used interchangeably herein with the term“an antibody which binds specifically to an antigen.”

As used herein, an antibody that “specifically binds to human CD40” isintended to refer to an antibody that binds to human CD40 protein (andpossibly a CD40 protein from one or more non-human species) but does notsubstantially bind to non-CD40 proteins. Preferably, the antibody bindsto human CD40 protein with “high affinity”, namely with a K_(D) of5.0×10⁻⁸ M or less, more preferably 1.0×10⁻⁸ M or less, and morepreferably 5.0×10⁻⁹ M or less.

The term “does not substantially bind” to a protein or cells, as usedherein, means does not bind or does not bind with a high affinity to theprotein or cells, i.e. binds to the protein or cells with a K_(D) of1.0×10⁻⁶ M or more, more preferably 1.0×10⁻⁵ M or more, more preferably1.0×10⁻⁴ M or more, more preferably 1.0×10⁻³ M or more, even morepreferably 1.0×10⁻² M or more.

The term “high affinity” for an IgG antibody refers to an antibodyhaving a K_(D) of 1.0×10⁻⁶ M or less, more preferably 5.0×10⁻⁸ M orless, even more preferably 1.0×10⁻⁸ M or less, even more preferably5.0×10⁻⁹ M or less and even more preferably 1.0×10⁻⁹ M or less for atarget antigen. However, “high affinity” binding can vary for otherantibody isotypes. For example, “high affinity” binding for an IgMisotype refers to an antibody having a K_(D) of 10⁻⁶ M or less, morepreferably 10⁻⁷ M or less, even more preferably 10⁻⁸ M or less.

The term “K_(assoc)” or “K_(a)”, as used herein, is intended to refer tothe association rate of a particular antibody-antigen interaction,whereas the term “K_(dis)” or “K_(d)”, as used herein, is intended torefer to the dissociation rate of a particular antibody-antigeninteraction. The term “K_(D)”, as used herein, is intended to refer tothe dissociation constant, which is obtained from the ratio of K_(d) toK_(a) (i.e., K_(d)/K_(a)) and is expressed as a molar concentration (M).K_(D) values for antibodies can be determined using methods wellestablished in the art. A preferred method for determining the K_(D) ofan antibody is by using surface plasmon resonance, preferably using abiosensor system such as a Biacore™ system.

The term “EC₅₀”, also known as half maximal effective concentration,refers to the concentration of an antibody which induces a responsehalfway between the baseline and maximum after a specified exposuretime.

The term “subject” includes any human or nonhuman animal. The term“nonhuman animal” includes all vertebrates, e.g., mammals andnon-mammals, such as non-human primates, sheep, dogs, cats, cows,horses, chickens, amphibians, and reptiles, although mammals arepreferred, such as non-human primates, sheep, dogs, cats, cows andhorses.

The term “agonistic CD40 antibody” or “agonistic anti-CD40 antibody”refers to an anti-CD40 antibody that binds to CD40 and activates orinduces CD40 signaling to promote immune cell activation andproliferation as well as cytokine and chemokine production. While theterm “antagonistic CD40 antibody” refers to an anti-CD40 antibody thatblocks or inhibits CD40 signaling that may be induced by CD40Lengagement.

The term “therapeutically effective amount” means an amount of theantibody of the present invention sufficient to prevent or amelioratethe symptoms associated with a disease or condition (such as a cancer)and/or lessen the severity of the disease or condition. Atherapeutically effective amount is understood to be in context to thecondition being treated, where the actual effective amount is readilydiscerned by those of skill in the art.

Various aspects of the invention are described in further detail in thefollowing subsections.

Anti-CD40 Antibodies Having Binding Specificity to Human CD40 andAdvantageous Functional Properties

Antibodies of the invention specifically bind to human CD40 with highaffinity, e.g., with a K_(D) of 1×10⁻⁸ M or less. The antibodies alsohave cross-reactivity with monkey CD40, but do not bind to mouse CD40.

The antibodies of the invention are agonistic CD40 antibodies thatactivate or induce CD40 signaling and thus involve in immune cellactivation and proliferation as well as cytokine and chemokineproduction.

The antibodies of the invention have in vivo anti-tumor effectcomparable to or better than prior art agnostic anti-CD40 antibodies,with equal or less toxicity. Tumors would not grow or even totallyvanish even after antibody administration has stopped.

Preferred antibodies of the invention are monoclonal antibodies.Additionally or alternatively, the antibodies can be, for example,mouse, chimeric or humanized monoclonal antibodies.

Monoclonal Anti-CD40 Antibody

A preferred antibody of the invention is the monoclonal antibodystructurally and chemically characterized as described below and in thefollowing Examples. The V_(H) amino acid sequence of the anti-CD40antibody is set forth in SEQ ID NOs: 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49 or 50. The V_(L) amino acid sequence of the anti-CD40antibody is shown in SEQ ID NOs: 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,or 61. The amino acid sequences of the heavy/light chain variableregions of the antibodies are summarized in Table 1 below, some clonessharing the same V_(H) or V_(L). Preferable amino acid sequence of theheavy chain constant region for all clones is set forth in SEQ ID NOs:62, 63 or 64, and preferbale amino acid sequence of the light chainconstant region for all clones is set forth in SEQ ID NOs: 65 or 66.

The V_(H) and V_(L) sequences (or CDR sequences) of other anti-CD40antibodies which bind to human CD40 can be “mixed and matched” with theV_(H) and V_(L) sequences (or CDR sequences) of the anti-CD40 antibodyof the present invention. Preferably, when V_(H) and V_(L) chains (orthe CDRs within such chains) are mixed and matched, a V_(H) sequencefrom a particular V_(H)/V_(L) pairing is replaced with a structurallysimilar V_(H) sequence. Likewise, preferably a V_(L) sequence from aparticular V_(H)/V_(L) pairing is replaced with a structurally similarV_(L) sequence.

Accordingly, in one embodiment, an antibody of the invention, or anantigen binding portion thereof, comprises:

(a) a heavy chain variable region comprising an amino acid sequencelisted above in Table 1; and

(b) a light chain variable region comprising an amino acid sequencelisted above in Table 1, or the V_(L) of another anti-CD40 antibody,wherein the antibody specifically binds human CD40.

In another embodiment, an antibody of the invention, or an antigenbinding portion thereof, comprises:

(a) the CDR1, CDR2, and CDR3 regions of the heavy chain variable regionlisted above in Table 1; and

(b) the CDR1, CDR2, and CDR3 regions of the light chain variable regionlisted above in Table 1 or the CDRs of another anti-CD40 antibody,wherein the antibody specifically binds human CD40.

In yet another embodiment, the antibody, or antigen binding portionthereof, includes the heavy chain variable CDR2 region of anti-CD40antibody combined with CDRs of other antibodies which bind human CD40,e.g., CDR1 and/or CDR3 from the heavy chain variable region, and/orCDR1, CDR2, and/or CDR3 from the light chain variable region of adifferent anti-CD40 antibody.

TABLE 1 Amino acid sequences of heavy/light chain variable regionsClone/SEQ HV- HV- HV- LV- LV- LV- ID NO. CDR1 CDR2 CDR3 HV CDR1 CDR2CDR3 LV 13A2 1 8 15 37 21 27 31 51 7B4 1 9 15 38 21 27 31 52 16A6 2 9 1539 21 27 31 53 29A10 3 10 16 40 22 28 32 54 92F6 4 11 17 41 23 29 33 5577D9 5 12 18 42 24 27 34 56 50F6 6 13 19 43 25 27 35 57 142F7 7 14 20 4426 30 36 58 13A2-VH0VL0 1 8 15 45 21 27 31 59 13A2-VH2VL2 1 8 15 46 2127 31 60 13A2-VH2VL3 1 8 15 46 21 27 31 61 13A2-VH3VL2 1 8 15 47 21 2731 60 13A2-VH3VL3 1 8 15 47 21 27 31 61 7B4-VH0VL0 1 9 15 48 21 27 31 597B4-VH2VL2 1 9 15 49 21 27 31 60 7B4-VH2VL3 1 9 15 49 21 27 31 617B4-VH3VL2 1 9 15 50 21 27 31 60 7B4-VH3VL3 1 9 15 50 21 27 31 61

In addition, it is well known in the art that the CDR3 domain,independently from the CDR1 and/or CDR2 domain(s), alone can determinethe binding specificity of an antibody for a cognate antigen and thatmultiple antibodies can predictably be generated having the same bindingspecificity based on a common CDR3 sequence. See, e.g., Klimka et al.,British J. of Cancer 83(2):252-260 (2000); Beiboer et al., J. Mol. Biol.296:833-849 (2000); Rader et al., Proc. Natl. Acad. Sci. U.S.A.95:8910-8915 (1998); Barbas et al., J. Am. Chem. Soc. 116:2161-2162(1994); Barbas et al., Proc. Natl. Acad. Sci. U.S.A. 92:2529-2533(1995); Ditzel et al., J. Immunol. 157:739-749 (1996); Berezov et al.,BIAjournal 8: Scientific Review 8 (2001); Igarashi et al., J. Biochem(Tokyo) 117:452-7 (1995); Bourgeois et al., J. Virol 72:807-10 (1998);Levi et al., Proc. Natl. Acad. Sci. U.S.A. 90:4374-8 (1993); Polymenisand Stoller, J. Immunol. 152:5218-5329 (1994) and Xu and Davis, Immunity13:37-45 (2000). See also, U.S. Pat. Nos. 6,951,646; 6,914,128;6,090,382; 6,818,216; 6,156,313; 6,827,925; 5,833,943; 5,762,905 and5,760,185. Each of these references is hereby incorporated by referencein its entirety.

Accordingly, in another embodiment, antibodies of the invention comprisethe CDR2 of the heavy chain variable region of the anti-CD40 antibodyand at least the CDR3 of the heavy and/or light chain variable region ofthe anti-CD40 antibody, or the CDR3 of the heavy and/or light chainvariable region of another anti-CD40 antibody, wherein the antibody iscapable of specifically binding to human CD40. These antibodiespreferably (a) compete for binding with CD40; (b) retain the functionalcharacteristics; (c) bind to the same epitope; and/or (d) have a similarbinding affinity as the anti-CD40 antibody of the present invention. Inyet another embodiment, the antibodies further may comprise the CDR2 ofthe light chain variable region of the anti-CD40 antibody, or the CDR2of the light chain variable region of another anti-CD40 antibody,wherein the antibody is capable of specifically binding to human CD40.In another embodiment, the antibodies of the invention may include theCDR1 of the heavy and/or light chain variable region of the anti-CD40antibody, or the CDR1 of the heavy and/or light chain variable region ofanother anti-CD40 antibody, wherein the antibody is capable ofspecifically binding to human CD40.

Conservative Modifications

In another embodiment, an antibody of the invention comprises a heavyand/or light chain variable region sequences of CDR1, CDR2 and CDR3sequences which differ from those of the anti-CD40 antibodies of thepresent invention by one or more conservative modifications. It isunderstood in the art that certain conservative sequence modificationcan be made which do not remove antigen binding. See, e.g., Brummell etal., (1993) Biochem 32:1180-8; de Wildt et al., (1997) Prot. Eng.10:835-41; Komissarov et al., (1997) J. Biol. Chem. 272:26864-26870;Hall et al., (1992) J. Immunol. 149:1605-12; Kelley and O'Connell (1993)Biochem. 32:6862-35; Adib-Conquy et al., (1998) Int. Immunol. 10:341-6and Beers et al., (2000) Clin. Can. Res. 6:2835-43.

Accordingly, in one embodiment, the antibody comprises a heavy chainvariable region comprising CDR1, CDR2, and CDR3 sequences and/or a lightchain variable region comprising CDR1, CDR2, and CDR3 sequences,wherein:

(a) the heavy chain variable region CDR1 sequence comprises a sequencelisted in Table 1 above, and/or conservative modifications thereof;and/or

(b) the heavy chain variable region CDR2 sequence comprises a sequencelisted in Table 1 above, and/or conservative modifications thereof;and/or

(c) the heavy chain variable region CDR3 sequence comprises a sequencelisted in Table 1 above, and conservative modifications thereof; and/or

(d) the light chain variable region CDR1, and/or CDR2, and/or CDR3sequences comprise the sequence(s) listed in Table 1 above; and/orconservative modifications thereof; and

(e) the antibody specifically binds human CD40.

The antibody of the present invention possesses one or more of thefollowing functional properties described above, such as high affinitybinding to human CD40, and the ability to induce ADCC or CDC againstCD40-expressing cells.

In various embodiments, the antibody can be, for example, a mouse,human, humanized or chimeric antibody.

As used herein, the term “conservative sequence modifications” isintended to refer to amino acid modifications that do not significantlyaffect or alter the binding characteristics of the antibody containingthe amino acid sequence. Such conservative modifications include aminoacid substitutions, additions and deletions. Modifications can beintroduced into an antibody of the invention by standard techniquesknown in the art, such as site-directed mutagenesis and PCR-mediatedmutagenesis. Conservative amino acid substitutions are ones in which theamino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine), beta-branchedside chains (e.g., threonine, valine, isoleucine) and aromatic sidechains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, oneor more amino acid residues within the CDR regions of an antibody of theinvention can be replaced with other amino acid residues from the sameside chain family and the altered antibody can be tested for retainedfunction (i.e., the functions set forth above) using the functionalassays described herein.

Engineered and Modified Antibodies

Antibodies of the invention can be prepared using an antibody having oneor more of the V_(H)/V_(L) sequences of the anti-CD40 antibody of thepresent invention as starting material to engineer a modified antibody.An antibody can be engineered by modifying one or more residues withinone or both variable regions (i.e., V_(H) and/or V_(L)), for examplewithin one or more CDR regions and/or within one or more frameworkregions. Additionally or alternatively, an antibody can be engineered bymodifying residues within the constant region(s), for example to alterthe effector function(s) of the antibody.

In certain embodiments, CDR grafting can be used to engineer variableregions of antibodies. Antibodies interact with target antigenspredominantly through amino acid residues that are located in the sixheavy and light chain complementarity determining regions (CDRs). Forthis reason, the amino acid sequences within CDRs are more diversebetween individual antibodies than sequences outside of CDRs. BecauseCDR sequences are responsible for most antibody-antigen interactions, itis possible to express recombinant antibodies that mimic the propertiesof specific naturally occurring antibodies by constructing expressionvectors that include CDR sequences from the specific naturally occurringantibody grafted onto framework sequences from a different antibody withdifferent properties (see, e.g., Riechmann et al., (1998) Nature332:323-327; Jones et al., (1986) Nature 321:522-525; Queen et al.,(1989) Proc. Natl. Acad. See also U.S.A. 86:10029-10033; U.S. Pat. Nos.5,225,539; 5,530,101; 5,585,089; 5,693,762 and 6,180,370).

Accordingly, another embodiment of the invention pertains to an isolatedmonoclonal antibody, or antigen binding portion thereof, comprising aheavy chain variable region comprising CDR1, CDR2, and CDR3 sequencescomprising the sequences of the present invention, as described above,and/or a light chain variable region comprising CDR1, CDR2, and CDR3sequences comprising the sequences of the present invention, asdescribed above. While these antibodies contain the V_(H) and V_(L) CDRsequences of the monoclonal antibody of the present invention, they cancontain different framework sequences.

Such framework sequences can be obtained from public DNA databases orpublished references that include germline antibody gene sequences. Forexample, germline DNA sequences for human heavy and light chain variableregion genes can be found in the “VBase” human germline sequencedatabase (available on the Internet at www.mrc-cpe.cam.ac.uk/vbase), aswell as in Kabat et al., (1991), cited supra; Tomlinson et al., (1992)J. Mol. Biol. 227:776-798; and Cox et al., (1994) Eur. J. Immunol.24:827-836; the contents of each of which are expressly incorporatedherein by reference. As another example, the germline DNA sequences forhuman heavy and light chain variable region genes can be found in theGenbank database. For example, the following heavy chain germlinesequences found in the HCo7 HuMAb mouse are available in theaccompanying Genbank Accession Nos.: 1-69 (NG-0010109, NT-024637 &BC070333), 3-33 (NG-0010109 & NT-024637) and 3-7 (NG-0010109 &NT-024637). As another example, the following heavy chain germlinesequences found in the HCo12 HuMAb mouse are available in theaccompanying Genbank Accession Nos.: 1-69 (NG-0010109, NT-024637 &BC070333), 5-51 (NG-0010109 & NT-024637), 4-34 (NG-0010109 & NT-024637),3-30.3 (CAJ556644) & 3-23 (AJ406678).

Antibody protein sequences are compared against a compiled proteinsequence database using one of the sequence similarity searching methodscalled the Gapped BLAST (Altschul et al., (1997), supra), which is wellknown to those skilled in the art.

Preferred framework sequences for use in the antibodies of the inventionare those that are structurally similar to the framework sequences usedby antibodies of the invention. The V_(H) CDR1, CDR2, and CDR3 sequencescan be grafted onto framework regions that have the identical sequenceas that found in the germline immunoglobulin gene from which theframework sequence derives, or the CDR sequences can be grafted ontoframework regions that contain one or more mutations as compared to thegermline sequences. For example, it has been found that in certaininstances it is beneficial to mutate residues within the frameworkregions to maintain or enhance the antigen binding ability of theantibody (see e.g., U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and6,180,370).

Another type of variable region modification is to mutate amino acidresidues within the V_(H) and/or V_(L) CDR1, CDR2 and/or CDR3 regions tothereby improve one or more binding properties (e.g., affinity) of theantibody of interest. Site-directed mutagenesis or PCR-mediatedmutagenesis can be performed to introduce the mutation(s) and the effecton antibody binding, or other functional property of interest, can beevaluated in in vitro or in vivo assays as known in the art. Preferablyconservative modifications (as known in the art) are introduced. Themutations can be amino acid substitutions, additions or deletions, butare preferably substitutions. Moreover, typically no more than one, two,three, four or five residues within a CDR region are altered.

Accordingly, in another embodiment, the invention provides isolatedanti-CD40 monoclonal antibodies, or antigen binding portions thereof,comprising a heavy chain variable region comprising: (a) a V_(H) CDR1region comprising the sequence of the present invention, or an aminoacid sequence having one, two, three, four or five amino acidsubstitutions, deletions or additions; (b) a V_(H) CDR2 regioncomprising the sequence of the present invention, or an amino acidsequence having one, two, three, four or five amino acid substitutions,deletions or additions; (c) a V_(H) CDR3 region comprising the sequenceof the present invention, or an amino acid sequence having one, two,three, four or five amino acid substitutions, deletions or additions;(d) a V_(L) CDR1 region comprising the sequence of the presentinvention, or an amino acid sequence having one, two, three, four orfive amino acid substitutions, deletions or additions; (e) a V_(L) CDR2region comprising the sequence of the present invention, or an aminoacid sequence having one, two, three, four or five amino acidsubstitutions, deletions or additions; and (f) a V_(L) CDR3 regioncomprising the sequence of the present invention, or an amino acidsequence having one, two, three, four or five amino acid substitutions,deletions or additions.

Engineered antibodies of the invention include those in whichmodifications have been made to framework residues within V_(H) and/orV_(L), e.g. to improve the properties of the antibody. Typically, suchframework modifications are made to decrease the immunogenicity of theantibody. For example, one approach is to “backmutate” one or moreframework residues to the corresponding germline sequence. Morespecifically, an antibody that has undergone somatic mutation cancontain framework residues that differ from the germline sequence fromwhich the antibody is derived. Such residues can be identified bycomparing the antibody framework sequences to the germline sequencesfrom which the antibody is derived.

Another type of framework modification involves mutating one or moreresidues within the framework region, or even within one or more CDRregions, to remove T cell epitopes to thereby reduce the potentialimmunogenicity of the antibody. This approach is also referred to as“deimmunization” and is described in further detail in U.S. PatentPublication No. 20030153043.

In addition, or as an alternative to modifications made within theframework or CDR regions, antibodies of the invention can be engineeredto include modifications within the Fc region, typically to alter one ormore functional properties of the antibody, such as serum half-life,complement fixation, Fc receptor binding, and/or antigen-dependentcellular cytotoxicity. Furthermore, an antibody of the invention can bechemically modified (e.g., one or more chemical moieties can be attachedto the antibody) or be modified to alter its glycosylation, again toalter one or more functional properties of the antibody.

In one embodiment, the hinge region of C_(H1) is modified in such thatthe number of cysteine residues in the hinge region is altered, e.g.,increased or decreased. This approach is described further in U.S. Pat.No. 5,677,425. The number of cysteine residues in the hinge region ofC_(H1) is altered to, for example, facilitate assembly of the light andheavy chains or to increase or decrease the stability of the antibody.

In another embodiment, the Fc hinge region of an antibody is mutated todecrease the biological half-life of the antibody. More specifically,one or more amino acid mutations are introduced into the C_(H1)-C_(H3)domain interface region of the Fc-hinge fragment such that the antibodyhas impaired Staphylococcyl protein A (SpA) binding relative to nativeFc-hinge domain SpA binding. This approach is described in furtherdetail in U.S. Pat. No. 6,165,745.

In still another embodiment, the glycosylation of an antibody ismodified. For example, a glycosylated antibody can be made (i.e., theantibody lacks glycosylation). Glycosylation can be altered to, forexample, increase the affinity of the antibody for antigen. Suchcarbohydrate modifications can be accomplished by, for example, alteringone or more sites of glycosylation within the antibody sequence. Forexample, one or more amino acid substitutions can be made that result inelimination of one or more variable region framework glycosylation sitesto thereby eliminate glycosylation at that site. Such aglycosylation mayincrease the affinity of the antibody for antigen. See, e.g., U.S. Pat.Nos. 5,714,350 and 6,350,861.

Additionally or alternatively, an antibody can be made that has analtered type of glycosylation, such as a hypofucosylated antibody havingreduced amounts of fucosyl residues or an antibody having increasedbisecting GlcNac structures. Such altered glycosylation patterns havebeen demonstrated to increase the ADCC ability of antibodies. Suchcarbohydrate modifications can be accomplished by, for example,expressing the antibody in a host cell with altered glycosylationmachinery. Cells with altered glycosylation machinery have beendescribed in the art and can be used as host cells in which to expressrecombinant antibodies of the invention to thereby produce an antibodywith altered glycosylation. For example, the cell lines Ms704, Ms705,and Ms709 lack the fucosyltransferase gene, FUT8(α(1,6)-fucosyltransferase), such that antibodies expressed in theMs704, Ms705, and Ms709 cell lines lack fucose on their carbohydrates.The Ms704, Ms705, and Ms709 FUT8−/− cell lines were created by thetargeted disruption of the FUT8 gene in CHO/DG44 cells using tworeplacement vectors (see U.S. Patent Publication No. 20040110704 andYamane-Ohnuki et al., (2004) Biotechnol Bioeng 87:614-22). As anotherexample, EP 1,176,195 describes a cell line with a functionallydisrupted FUT8 gene, which encodes a fucosyl transferase, such thatantibodies expressed in such a cell line exhibit hypofucosylation byreducing or eliminating the α-1,6 bond-related enzyme. EP 1,176,195 alsodescribes cell lines which have a low enzyme activity for adding fucoseto the N-acetylglucosamine that binds to the Fc region of the antibodyor does not have the enzyme activity, for example the rat myeloma cellline YB2/0 (ATCC CRL 1662). PCT Publication WO 03/035835 describes avariant CHO cell line, Lec13 cells, with reduced ability to attachfucose to Asn(297)-linked carbohydrates, also resulting inhypofucosylation of antibodies expressed in that host cell (see alsoShields et al., (2002) J. Biol. Chem. 277:26733-26740). Antibodies witha modified glycosylation profile can also be produced in chicken eggs,as described in PCT Publication WO 06/089231. Alternatively, antibodieswith a modified glycosylation profile can be produced in plant cells,such as Lemna. Methods for production of antibodies in a plant systemare disclosed in the U.S. patent application No. 60/836,998corresponding to Alston & Bird LLP attorney docket No. 040989/314911,filed on Aug. 11, 2006. PCT Publication WO 99/54342 describes cell linesengineered to express glycoprotein-modifying glycosyl transferases(e.g., β(1,4)-N-acetylglucosaminyltransferase III (GnTIII)) such thatantibodies expressed in the engineered cell lines exhibit increasedbisecting GlcNac structures which results in increased ADCC activity ofthe antibodies (see also Umana et al., (1999) Nat. Biotech. 17:176-180).Alternatively, the fucose residues of the antibody can be cleaved offusing a fucosidase enzyme; e.g., the fucosidase α-L-fucosidase removesfucosyl residues from antibodies (Tarentino et al., (1975) Biochem.14:5516-23).

Another modification of the antibodies herein that is contemplated bythis disclosure is pegylation. An antibody can be pegylated to, forexample, increase the biological (e.g., serum) half-life of theantibody. To pegylate an antibody, the antibody, or fragment thereof,typically is reacted with polyethylene glycol (PEG), such as a reactiveester or aldehyde derivative of PEG, under conditions in which one ormore PEG groups become attached to the antibody or antibody fragment.Preferably, the pegylation is carried out via an acylation reaction oran alkylation reaction with a reactive PEG molecule (or an analogousreactive water-soluble polymer). As used herein, the term “polyethyleneglycol” is intended to encompass any of the forms of PEG that have beenused to derivatize other proteins, such as mono (C₁-C₁₀) alkoxy- oraryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certainembodiments, the antibody to be pegylated is an aglycosylated antibody.Methods for pegylating proteins are known in the art and can be appliedto the antibodies of the invention. See, e.g., EPO 154 316 and EP 0 401384.

Antibody's Physical Properties

Antibodies of the invention can be characterized by their variousphysical properties, to detect and/or differentiate different classesthereof.

For example, antibodies can contain one or more glycosylation sites ineither the light or heavy chain variable region. Such glycosylationsites may result in increased immunogenicity of the antibody or analteration of the pK of the antibody due to altered antigen binding(Marshall et al (1972) Annu Rev Biochem 41:673-702; Gala and Morrison(2004) J Immunol 172:5489-94; Wallick et al (1988) J Exp Med168:1099-109; Spiro (2002) Glycobiology 12:43R-56R; Parekh et al (1985)Nature 316:452-7; Mimura et al., (2000) Mol Immunol 37:697-706).Glycosylation has been known to occur at motifs containing an N—X—S/Tsequence. In some instances, it is preferred to have an anti-CD40antibody that does not contain variable region glycosylation. This canbe achieved either by selecting antibodies that do not contain theglycosylation motif in the variable region or by mutating residueswithin the glycosylation region.

In a preferred embodiment, the antibodies do not contain asparagineisomerism sites. The deamidation of asparagine may occur on N-G or D-Gsequences and result in the creation of an isoaspartic acid residue thatintroduces a kink into the polypeptide chain and decreases its stability(isoaspartic acid effect).

Each antibody will have a unique isoelectric point (pI), which generallyfalls in the pH range between 6 and 9.5. The pI for an IgG1 antibodytypically falls within the pH range of 7-9.5 and the pI for an IgG4antibody typically falls within the pH range of 6-8. There isspeculation that antibodies with a pI outside the normal range may havesome unfolding and instability under in vivo conditions. Thus, it ispreferred to have an anti-CD40 antibody that contains a pI value thatfalls in the normal range. This can be achieved either by selectingantibodies with a pI in the normal range or by mutating charged surfaceresidues.

Nucleic Acid Molecules Encoding Antibodies of the Invention

In another aspect, the invention provides nucleic acid molecules thatencode heavy and/or light chain variable regions, or CDRs, of theantibodies of the invention. The nucleic acids can be present in wholecells, in a cell lysate, or in a partially purified or substantiallypure form. A nucleic acid is “isolated” or “rendered substantially pure”when purified away from other cellular components or other contaminants,e.g., other cellular nucleic acids or proteins, by standard techniques.A nucleic acid of the invention can be, e.g., DNA or RNA and may or maynot contain intronic sequences. In a preferred embodiment, the nucleicacid is a cDNA molecule.

Nucleic acids of the invention can be obtained using standard molecularbiology techniques. For antibodies expressed by hybridomas (e.g.,hybridomas prepared from transgenic mice carrying human immunoglobulingenes as described further below), cDNAs encoding the light and heavychains of the antibody made by the hybridoma can be obtained by standardPCR amplification or cDNA cloning techniques. For antibodies obtainedfrom an immunoglobulin gene library (e.g., using phage displaytechniques), a nucleic acid encoding such antibodies can be recoveredfrom the gene library.

Preferred nucleic acids molecules of the invention include thoseencoding the V_(H) and V_(L) sequences of the CD40 monoclonal antibodyor the CDRs. Once DNA fragments encoding V_(H) and V_(L) segments areobtained, these DNA fragments can be further manipulated by standardrecombinant DNA techniques, for example to convert the variable regiongenes to full-length antibody chain genes, to Fab fragment genes or to ascFv gene. In these manipulations, a V_(L)- or V_(H)-encoding DNAfragment is operatively linked to another DNA fragment encoding anotherprotein, such as an antibody constant region or a flexible linker. Theterm “operatively linked”, as used in this context, is intended to meanthat the two DNA fragments are joined such that the amino acid sequencesencoded by the two DNA fragments remain in-frame.

The isolated DNA encoding the V_(H) region can be converted to afull-length heavy chain gene by operatively linking the V_(H)-encodingDNA to another DNA molecule encoding heavy chain constant regions(C_(H1), C_(H2) and C_(H3)). The sequences of human heavy chain constantregion genes are known in the art and DNA fragments encompassing theseregions can be obtained by standard PCR amplification. The heavy chainconstant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgDconstant region, but most preferably is an IgG1 or IgG4 constant region.For a Fab fragment heavy chain gene, the V_(H)-encoding DNA can beoperatively linked to another DNA molecule encoding only the heavy chainC_(H1) constant region.

The isolated DNA encoding the V_(L) region can be converted to afull-length light chain gene (as well as a Fab light chain gene) byoperatively linking the V_(L)-encoding DNA to another DNA moleculeencoding the light chain constant region, C_(L). The sequences of humanlight chain constant region genes are known in the art and DNA fragmentsencompassing these regions can be obtained by standard PCRamplification. In preferred embodiments, the light chain constant regioncan be a kappa or lambda constant region.

To create a scFv gene, the V_(H)- and V_(L)-encoding DNA fragments areoperatively linked to another fragment encoding a flexible linker, e.g.,encoding the amino acid sequence (Gly4-Ser)3, such that the V_(H) andV_(L) sequences can be expressed as a contiguous single-chain protein,with the V_(L) and V_(H) regions joined by the flexible linker (seee.g., Bird et al., (1988) Science 242:423-426; Huston et al., (1988)Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., (1990)Nature 348:552-554).

Production of Monoclonal Antibodies of the Invention

Monoclonal antibodies (mAbs) of the present invention can be producedusing the well-known somatic cell hybridization (hybridoma) technique ofKohler and Milstein (1975) Nature 256: 495. Other embodiments forproducing monoclonal antibodies include viral or oncogenictransformation of B lymphocytes and phage display techniques. Chimericor humanized antibodies are also well known in the art. See e.g., U.S.Pat. Nos. 4,816,567; 5,225,539; 5,530,101; 5,585,089; 5,693,762 and6,180,370, the contents of which are specifically incorporated herein byreference in their entirety.

Generation of Transfectomas Producing Monoclonal Antibodies of theInvention

Antibodies of the invention also can be produced in a host celltransfectoma using, for example, a combination of recombinant DNAtechniques and gene transfection methods as is well known in the art(e.g., Morrison, S. (1985) Science 229:1202). In one embodiment, DNAencoding partial or full-length light and heavy chains obtained bystandard molecular biology techniques is inserted into one or moreexpression vectors such that the genes are operatively linked totranscriptional and translational regulatory sequences. In this context,the term “operatively linked” is intended to mean that an antibody geneis ligated into a vector such that transcriptional and translationalcontrol sequences within the vector serve their intended function ofregulating the transcription and translation of the antibody gene.

The term “regulatory sequence” is intended to include promoters,enhancers and other expression control elements (e.g., polyadenylationsignals) that control the transcription or translation of the antibodygenes. Such regulatory sequences are described, e.g., in Goeddel (GeneExpression Technology. Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990)). Preferred regulatory sequences for mammalian hostcell expression include viral elements that direct high levels ofprotein expression in mammalian cells, such as promoters and/orenhancers derived from cytomegalovirus (CMV), Simian Virus 40 (SV40),adenovirus, e.g., the adenovirus major late promoter (AdMLP) andpolyoma. Alternatively, nonviral regulatory sequences can be used, suchas the ubiquitin promoter or β-globin promoter. Still further,regulatory elements composed of sequences from different sources, suchas the SRα promoter system, which contains sequences from the SV40 earlypromoter and the long terminal repeat of human T cell leukemia virustype 1 (Takebe et al., (1988) Mol. Cell. Biol. 8:466-472). Theexpression vector and expression control sequences are chosen to becompatible with the expression host cell used.

The antibody light chain gene and the antibody heavy chain gene can beinserted into the same or separate expression vectors. In preferredembodiments, the variable regions are used to create full-lengthantibody genes of any antibody isotype by inserting them into expressionvectors already encoding heavy chain constant and light chain constantregions of the desired isotype such that the V_(H) segment isoperatively linked to the C_(H) segment(s) within the vector and theV_(L) segment is operatively linked to the C_(L) segment within thevector. Additionally or alternatively, the recombinant expression vectorcan encode a signal peptide that facilitates secretion of the antibodychain from a host cell. The antibody chain gene can be cloned into thevector such that the signal peptide is linked in-frame to the aminoterminus of the antibody chain gene. The signal peptide can be animmunoglobulin signal peptide or a heterologous signal peptide (i.e., asignal peptide from a non-immunoglobulin protein).

In addition to the antibody chain genes and regulatory sequences, therecombinant expression vectors of the invention can carry additionalsequences, such as sequences that regulate replication of the vector inhost cells (e.g., origins of replication) and selectable marker genes.The selectable marker gene facilitates selection of host cells intowhich the vector has been introduced (see, e.g., U.S. Pat. Nos.4,399,216; 4,634,665 and 5,179,017). For example, typically theselectable marker gene confers resistance to drugs, such as G418,hygromycin or methotrexate, on a host cell into which the vector hasbeen introduced. Preferred selectable marker genes include thedihydrofolate reductase (DHFR) gene (for use in dhfr-host cells withmethotrexate selection/amplification) and the neo gene (for G418selection).

For expression of the light and heavy chains, the expression vector(s)encoding the heavy and light chains is transfected into a host cell bystandard techniques. The various forms of the term “transfection” areintended to encompass a wide variety of techniques commonly used for theintroduction of exogenous DNA into a prokaryotic or eukaryotic hostcell, e.g., electroporation, calcium-phosphate precipitation,DEAE-dextran transfection and the like. Although it is theoreticallypossible to express the antibodies of the invention in eitherprokaryotic or eukaryotic host cells, expression of antibodies ineukaryotic cells, and most preferably mammalian host cells, is the mostpreferred because such eukaryotic cells, and in particular mammaliancells, are more likely than prokaryotic cells to assemble and secrete aproperly folded and immunologically active antibody.

Preferred mammalian host cells for expressing the recombinant antibodiesof the invention include Chinese Hamster Ovary (CHO cells) (includingdhfr-CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad.Sci. USA 77:4216-4220, used with a DHFR selectable marker, e.g., asdescribed in R. J. Kaufman and P. A. Sharp (1982) J. Mol. Biol.159:601-621), NSO myeloma cells, COS cells and SP2 cells. In particularfor use with NSO myeloma cells, another preferred expression system isthe GS gene expression system disclosed in WO 87/04462, WO 89/01036 andEP 338,841. When recombinant expression vectors encoding antibody genesare introduced into mammalian host cells, the antibodies are produced byculturing the host cells for a period of time sufficient to allow forexpression of the antibody in the host cells or, more preferably,secretion of the antibody into the culture medium in which the hostcells are grown. Antibodies can be recovered from the culture mediumusing standard protein purification methods.

Immunoconjugates

Antibodies of the invention can be conjugated to a therapeutic agent toform an immunoconjugate such as an antibody-drug conjugate (ADC).Suitable therapeutic agents include cytotoxins, alkylating agents, DNAminor groove binders, DNA intercalators, DNA crosslinkers, histonedeacetylase inhibitors, nuclear export inhibitors, proteasomeinhibitors, topoisomerase I or II inhibitors, heat shock proteininhibitors, tyrosine kinase inhibitors, antibiotics, and anti-mitoticagents. In the ADC, the antibody and therapeutic agent preferably areconjugated via a linker cleavable such as a peptidyl, disulfide, orhydrazone linker. More preferably, the linker is a peptidyl linker suchas Val-Cit, Ala-Val, Val-Ala-Val, Lys-Lys, Pro-Val-Gly-Val-Val,Ala-Asn-Val, Val-Leu-Lys, Ala-Ala-Asn, Cit-Cit, Val-Lys, Lys, Cit, Ser,or Glu. The ADCs can be prepared as described in U.S. Pat. Nos.7,087,600; 6,989,452; and 7,129,261; PCT Publications WO 02/096910; WO07/038,658; WO 07/051,081; WO 07/059,404; WO 08/083,312; and WO08/103,693; U.S. Patent Publications 20060024317; 20060004081; and20060247295; the disclosures of which are incorporated herein byreference.

Bispecific Molecules

In another aspect, the present disclosure features bispecific moleculescomprising one or more antibodies of the invention linked to at leastone other functional molecule, e.g., another peptide or protein (e.g.,another antibody or ligand for a receptor) to generate a bispecificmolecule that binds to at least two different binding sites or targetmolecules. Thus, as used herein, “bispecific molecule” includesmolecules that have three or more specificities.

In an embodiment, a bispecific molecule has, in addition to an anti-Fcbinding specificity and an anti-CD40 binding specificity, a thirdspecificity. The third specificity can be for an anti-enhancement factor(EF), e.g., a molecule that binds to a surface protein involved incytotoxic activity and thereby increases the immune response against thetarget cell. For example, the anti-enhancement factor can bind acytotoxic T-cell (e.g. via CD2, CD3, CD8, CD28, CD4, CD40, or ICAM-1) orother immune cell, resulting in an increased immune response against thetarget cell.

Bispecific molecules may be in many different formats and sizes. At oneend of the size spectrum, a bispecific molecule retains the traditionalantibody format, except that, instead of having two binding arms ofidentical specificity, it has two binding arms each having a differentspecificity. At the other extreme are bispecific molecules consisting oftwo single-chain antibody fragments (scFv's) linked by a peptide chain,a so-called Bs(scFv) 2 construct. Intermediate-sized bispecificmolecules include two different F(ab) fragments linked by a peptidyllinker. Bispecific molecules of these and other formats can be preparedby genetic engineering, somatic hybridization, or chemical methods. See,e.g., Kufer et al, cited supra; Cao and Suresh, Bioconjugate Chemistry,9 (6), 635-644 (1998); and van Spriel et al., Immunology Today, 21 (8),391-397 (2000), and the references cited therein.

Antibody-Encoding or Antibody-Bearing Oncolytic Virus

An oncolytic virus preferabtially infects and kills cancer cells.Antibodies of the present invention can be used in conjunction withoncolytic viruses. Alternatively, oncolytic viruses encoding antibodiesof the present invention can be introduced into human body.

Pharmaceutical Compositions

In another aspect, the present disclosure provides a pharmaceuticalcomposition comprising one or more antibodies of the present inventionformulated together with a pharmaceutically acceptable carrier. Thecomposition may optionally contain one or more additionalpharmaceutically active ingredients, such as another antibody or a drug,such as anti-VISTA antibody. The pharmaceutical compositions of theinvention also can be administered in a combination therapy with, forexample, another anti-cancer agent, another anti-inflammatory agent, oran antimicrobial agent.

The pharmaceutical composition can comprise any number of excipients.Excipients that can be used include carriers, surface active agents,thickening or emulsifying agents, solid binders, dispersion orsuspension aids, solubilizers, colorants, flavoring agents, coatings,disintegrating agents, lubricants, sweeteners, preservatives, isotonicagents, and combinations thereof. The selection and use of suitableexcipients is taught in Gennaro, ed., Remington: The Science andPractice of Pharmacy, 20th Ed. (Lippincott Williams & Wilkins 2003), thedisclosure of which is incorporated herein by reference.

Preferably, the pharmaceutical composition is suitable for intravenous,intramuscular, subcutaneous, parenteral, spinal or epidermaladministration (e.g., by injection or infusion). Depending on the routeof administration, the active ingredient can be coated in a material toprotect it from the action of acids and other natural conditions thatmay inactivate it. The phrase “parenteral administration” as used hereinmeans modes of administration other than enteral and topicaladministration, usually by injection, and includes, without limitation,intravenous, intramuscular, intraarterial, intrathecal, intracapsular,intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal,subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid,intraspinal, epidural and intrasternal injection and infusion.Alternatively, an antibody of the invention can be administered via anon-parenteral route, such as a topical, epidermal or mucosal route ofadministration, e.g., intranasally, orally, vaginally, rectally,sublingually or topically.

Pharmaceutical compositions can be in the form of sterile aqueoussolutions or dispersions. They can also be formulated in amicroemulsion, liposome, or other ordered structure suitable to highdrug concentration.

The amount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will vary depending upon thesubject being treated and the particular mode of administration and willgenerally be that amount of the composition which produces a therapeuticeffect. Generally, out of one hundred percent, this amount will rangefrom about 0.01% to about ninety-nine percent of active ingredient,preferably from about 0.1% to about 70%, most preferably from about 1%to about 30% of active ingredient in combination with a pharmaceuticallyacceptable carrier.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus can beadministered, several divided doses can be administered over time or thedose can be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. It is especially advantageousto formulate parenteral compositions in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form as used hereinrefers to physically discrete units suited as unitary dosages for thesubjects to be treated; each unit contains a predetermined quantity ofactive ingredient calculated to produce the desired therapeutic effectin association with the required pharmaceutical carrier. Alternatively,antibody can be administered as a sustained release formulation, inwhich case less frequent administration is required.

For administration of the antibody, the dosage may range from about0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the host bodyweight. For example dosages can be 0.3 mg/kg body weight, 1 mg/kg bodyweight, 3 mg/kg body weight, 5 mg/kg body weight or 10 mg/kg body weightor within the range of 1-10 mg/kg. An exemplary treatment regime entailsadministration once per week, once every two weeks, once every threeweeks, once every four weeks, once a month, once every 3 months or onceevery three to 6 months. Preferred dosage regimens for an anti-CD40antibody of the invention include 1 mg/kg body weight or 3 mg/kg bodyweight via intravenous administration, with the antibody being givenusing one of the following dosing schedules: (i) every four weeks forsix dosages, then every three months; (ii) every three weeks; (iii) 3mg/kg body weight once followed by 1 mg/kg body weight every threeweeks. In some methods, dosage is adjusted to achieve a plasma antibodyconcentration of about 1-1000 μg/ml and in some methods about 25-300μg/ml.

A “therapeutically effective dosage” of an anti-CD40 antibody of theinvention preferably results in a decrease in severity of diseasesymptoms, an increase in frequency and duration of disease symptom-freeperiods, or a prevention of impairment or disability due to the diseaseaffliction. For example, for the treatment of tumor-bearing subjects, a“therapeutically effective dosage” preferably inhibits tumor growth byat least about 20%, more preferably by at least about 40%, even morepreferably by at least about 60%, and still more preferably by at leastabout 80% relative to untreated subjects. A therapeutically effectiveamount of a therapeutic antibody can decrease tumor size, or otherwiseameliorate symptoms in a subject, which is typically a human or can beanother mammal.

The pharmaceutical composition can be a controlled release formulation,including implants, transdermal patches, and microencapsulated deliverysystems. Biodegradable, biocompatible polymers can be used, such asethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. See, e.g., Sustained andControlled Release Drug Delivery Systems, J. R. Robinson, ed., MarcelDekker, Inc., New York, 1978.

Therapeutic compositions can be administered via medical devices such as(1) needleless hypodermic injection devices (e.g., U.S. Pat. Nos.5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; and4,596,556); (2) micro-infusion pumps (U.S. Pat. No. 4,487,603); (3)transdermal devices (U.S. Pat. No. 4,486,194); (4) infusion apparatuses(U.S. Pat. Nos. 4,447,233 and 4,447,224); and (5) osmotic devices (U.S.Pat. Nos. 4,439,196 and 4,475,196); the disclosures of which areincorporated herein by reference.

In certain embodiments, the monoclonal antibodies of the invention canbe formulated to ensure proper distribution in vivo. For example, toensure that the therapeutic antibody of the invention cross theblood-brain barrier, they can be formulated in liposomes, which mayadditionally comprise targeting moieties to enhance selective transportto specific cells or organs. See, e.g. U.S. Pat. Nos. 4,522,811;5,374,548; 5,416,016; and 5,399,331; V. V. Ranade (1989) J. Clin.Pharmacol. 29:685; Umezawa et al., (1988) Biochem. Biophys. Res. Commun.153:1038; Bloeman et al., (1995) FEBS Lett. 357:140; M. Owais et al.,(1995) Antimicrob. Agents Chemother. 39:180; Briscoe et al., (1995) Am.J. Physiol. 1233:134; Schreier et al., (1994) J. Biol. Chem. 269:9090;Keinanen and Laukkanen (1994) FEBS Lett 346:123; and Killion and Fidler(1994) Immunomethods 4:273.

Uses and Methods of the Invention

Antibodies (compositions, bispecifics, and immunoconjugates) of thepresent invention have numerous in vitro and in vivo utilitiesinvolving, for example, treatment and/or prevention of cancers,inflammatory diseases, or infectious diseases. The antibodies can beadministered to human subjects, e.g., in vivo, to inhibit tumor growth.

Given the ability of anti-CD40 antibodies of the invention to inhibitproliferation and survival of cancer cells, the invention providesmethods for inhibiting growth of tumor cells in a subject comprisingadministering to the subject an antibody of the invention such thatgrowth of the tumor is inhibited in the subject. Non-limiting examplesof tumors that can be treated by antibodies of the invention include,but not limited to, B cell lymphoma, chronic lymphocytic leukemia,multiple myeloma, melanoma, colon adenocarcinoma, pancreas cancer, coloncancer, gastric intestine cancer, prostate cancer, bladder cancer,kidney cancer, ovary cancer, cervix cancer, breast cancer, lung cancer,and nasopharynx cancer, original and/or metastatic. Additionally,refractory or recurrent malignancies whose growth may be inhibited usingthe antibodies of the invention.

In another aspect, the invention provides a method for treating aninflammatory disease, an infectious disease, atherothrombosis, or arespiratory disease in a subject, comprising administering to thesubject a therapeutically effective amount of the antibody, orantigen-binding portion thereof, of the invention. Additionalanti-inflammatory agents, antimicrobial agents or other therapeticalagents can be administered with the antibody, or an antigen-bindingportion thereof, of the invention.

Generally speaking, the antibodies of the invention can be used toenhance an immune response in a subject.

These and other methods of the invention are discussed in further detailbelow.

Combination Therapy

In another aspect, the invention provides methods of combination therapyin which an anti-CD40 antibody (or antigen-binding portion thereof) ofthe present invention is co-administered with one or more additionalantibodies that are effective in inhibiting tumor growth in a subject.In one embodiment, the invention provides a method for inhibiting tumorgrowth in a subject comprising administering to the subject an anti-CD40antibody and one or more additional antibodies, such as an anti-VISTAantibody, an anti-LAG-3 antibody, an anti-PD-L1 antibody, and anti-PD-1antibody and/or an anti-CTLA-4 antibody. In certain embodiments, thesubject is human.

The CD40 signaling activation can also be further combined with standardcancer treatments. For example, CD40 signaling activationa can becombined with CTLA-4 and/or LAG-3 and/or PD-1 blockade and alsochemotherapeutic regimes. For example, a chemotherapeutic agent can beadministered with the anti-CD40 antibodies, which may be a cytotoxicagent. For example, epitubicin, oxaliplatin, and 5-FU are administeredto patients receiving anti-CD40 therapy.

Optionally, the combination of anti-CD40 and one or more additionalantibodies (e.g., anti-CTLA-4 and/or anti-LAG-3 and/or anti-PD-1antibodies) can be further combined with an immunogenic agent, such ascancerous cells, purified tumor antigens (including recombinantproteins, peptides, and carbohydrate molecules), and cells transfectedwith genes encoding immune stimulating cytokines (He et al., (2004) J.Immunol. 173:4919-28). Non-limiting examples of tumor vaccines that canbe used include peptides of melanoma antigens, such as peptides ofgp100, MAGE antigens, Trp-2, MARTI_ and/or tyrosinase, or tumor cellstransfected to express the cytokine GM-CSF.

Other therapies that may be combined with anti-CD40 antibody includes,but not limited to, interleukin-2 (IL-2) administration, radiation,surgery, or hormone deprivation.

The combination of therapeutic agents discussed herein can beadministered concurrently as a single composition in a pharmaceuticallyacceptable carrier, or concurrently as separate compositions with eachagent in a pharmaceutically acceptable carrier. In another embodiment,the combination of therapeutic agents can be administered sequentially.

Furthermore, if more than one dose of the combination therapy isadministered sequentially, the order of the sequential administrationcan be reversed or kept in the same order at each time point ofadministration, sequential administrations can be combined withconcurrent administrations, or any combination thereof.

The present disclosure is further illustrated by the following examples,which should not be construed as further limiting. The contents of allfigures and all references, Genbank sequences, patents and publishedpatent applications cited throughout this application are expresslyincorporated herein by reference.

EXAMPLES Example 1 Construction of HEK293A Cell Lines Stably ExpressingHuman, Rhesus or Mouse CD40

Stable cell lines overexpressing human, rhesus or mouse CD40 wereconstructed using HEK293A cells (Cobioer, NJ, China). Briefly, human,rhesus or mouse CD40 cDNA sequence (SEQ ID NOs: 67, 69 and 71, encodingamino acid sequences set forth in SEQ ID NOs: 68, 70 and 72,respectively) were synthesized, and then subcloned intopLV-EGFP(2A)-Puro vectors. Lentiviruses were generated in HEK-293T cells(Cobioer, NJ, China) by cotransfection of pLV-EGFP(2A)-Puro-CD40, psPAXand pMD2.G plasmids, according to the instruction in Lipofectamine 3000kit (Thermo Fisher Scientific, US). Three days post cotransfection, thelentiviruses were harvested from the cell culture medium (DMEM medium(Cat #: SH30022.01, Gibco) with 10% FBS (Cat #: FND500, Excell)) ofrespective HEK-293T cells. Finally, HEK293A cells were infected with thelentiviruses to generate HEK293A cell lines stably expressing human,rhesus or mouse CD40, namely HEK293A/humanCD40, HEK293A/rhesusCD40 orHEK293A/mouseCD40 cells. Transfected HEK293A cells were then cultured inmedium (DMEM+10% FBS) containing 0.2 μg/ml puromycin (Cat #: A11138-03,Gibco) for 7 days. The expression of human CD40 and rhesus CD40 wereconfirmed by FACS using a commercially avaibale anti-human CD40 antibody(PE-anti-human CD40, Biolegend, US, Cat #: 313006). Similarly, theexpression of mouse CD40 was confirmed by FACS using a commerciallyavaibale anti-mouse CD40 antibody (PE-anti-mouse CD40, Biolegend, US,Cat #: 124609).

Example 2 Generation of Hybridoma Cell Lines Producing Monoclonal MouseAntibodies Against Human CD40

Murine anti-human CD40 monoclonal antibodies (mAbs) were generated usingthe conventional hybridoma fusion technology with some modifications.

Immunization

Thirteen BALB/c mice (Beijing Vital River Laboratory Animal TechnologyCo., Ltd, Beijing, China) were injected with recombinant human CD40(ECD)-his (Sino Biological, CN, Cat #: 10774-H08H) and/or recombinantrhesus CD40 (ECD)-hFc (Sino Biological, CN, Cat #:90097-C02H) followingthe scheme in Table 2 below. The human CD40 (ECD)-his and rhesus CD40(ECD)-hFc were emulsified by sonication with an equal volume of CompleteFreund's Adjuvant (SIGMA, USA, Cat #: F5881-10*10 ML), IncompleteFreund's Adjuvant (SIGMA, USA, Cat #: F5506-6*10 ML), or PBS.

TABLE 2 Immunization scheme Primary 1st Boost 2nd Boost 3rd Boost FinalBoost Day 0 14 28 42 56 Protein and Human Human Human Human Human doseCD40 CD40 CD40 CD40 CD40 (ECD)-his (ECD)-his (ECD)-his (ECD)-his(ECD)-his (50 μg/mouse) (25 μg/mouse) (25 μg/mouse) (25 μg/mouse) (50μg/mouse) + + Rhesus Rhesus CD40(ECD)-hFc CD40(ECD)-hFc (25 μg/mouse)(25 μg/mouse) Adjuvant Complete Incomplete Incomplete Incomplete PBSFreund's Freund's Freund's Freund's Way of i.p. s.c. i.p. s.c. i.p.immunization

One week after each boost, 50 μl of murine serum was collected from eachmouse for titer determination by ELISA using the recombinant humanCD40(ECD)-hFc (Sino Biological, CN, Cat #:10774-H02H) and rhesus CD40(ECD)-hFc (Sino Biological, CN, Cat #: 90097-C02H). Titer determinationwas also done by FACS using HEK293A overexpressing human CD40, rhesusCD40 or mouse CD40 as prepared in Example 1. Based on the ELISA and FACSanalysis results after the final boost, seven mice with highest serumtiters were chosen for hybridoma cell line generation.

Generation of Hybridoma Cell Lines

Hybridoma cell lines were generated using the conventional hybridomafusion technology with minor modifications.

Four days after the final boost, mice were sacrificed, and spleens werecollected and prepared as single cell suspensions in PBS. Thespleenocytes were washed for three times with DMEM medium (Hyclone, Cat#: SH30243.01B). Viable myeloma cells SP2/0 (ATCC, CRL-1581) at thelog-phase were mixed with the murine spleenocytes in a ratio of 1:4. Thecells were then washed 2 times and then cell fusion was performed withPEG (Sigma, Cat #: P7181). The post-fusion cells were washed with DMEMmedium for three times and suspended in cell growth media (RPMI medium1640 (Gibco, Cat #:C22400500CP)) supplemented with 10% FBS and 1×HAT(Sigma, H0262). The cell suspension was plated into 96 well cell cultureplates, 200 μl per well (5×10⁴ cells/well), and incubated in a 37° C.humidified 5% CO₂ incubator for 7 days. Then, the growth media wasreplaced by fresh growth media supplemented with 10% FBS+1× HT (Sigma,H0137). 2˜3 days later, hybridoma cells were screened by ELISA and FACS.

Screening of Hybridoma Cell Lines by ELISA

High-throughput ELISA binding assay was firstly used to screen forhybridoma clones producing monoclonal antibodies binding to human CD40.Hybridoma clones producing monoclonal antibodies binding to human CD40were further tested for their ability to cross-react with rhesus ormouse CD40.

For ELISA assays, 96-well ELISA plates were coated with 100 μl/wellhuman CD40 (ECD)-his (0.5 μg/ml, Sino Biological, CN, Cat #:10774-H08H),rhesus CD40 (ECD)-hFc (0.5 μg/ml, Sino Biological, CN, Cat #:90097-C02H) or murine CD40-His (0.5 μg/ml, Sino Biological, CN, Cat #:50324-M03H) at room temperature overnight. Plates were washed 3 timeswith PBST buffer (PBS+0.05% Tween 20) and blocked with 200 μl ofblocking buffer (PBS containing 1% BSA, 1% goat serum, and 0.05% Tween20) at RT for 2 hr and washed for 3 times with PBST. Then, hybridomacell culture supernatant was diluted 10× with dilution buffer (PBScontaining 1% BSA, 1% goat serum, and 0.01% Tween 20) and added to theplates, 100 μl per well. After incubated at RT for 1 hr, plates werewashed 3 times with PBST and then 100 μl of goat anti-mouse Fc-HRP(1:5000, Sigma, US, Cat #:A9309-1 ml) was added to each well. Afterincubated at RT for 1 hr, plates were washed 3 times with PBST and then80 μl of TMB was added to each well. Five to ten minutes later, 80 μl of0.16 M sulfuric acid was added to each well and then OD450 was read onSpectraMaxR i3X (Molecular Devies, US).

With the ELISA assays, 234 hybridoma clones were identified to havespecific binding to both human and rhesus monkey CD40.

Screening of Hybridoma Cell Lines by FACS

The 234 hybridoma clones were further screened for their bindingcapacity to human, rhesus or mouse CD40 expressed on HEK293A cells.Briefly, 100,000 HEK293A/human CD40 cells, HEK293A/rhesusCD40 cells orHEK293A/mouseCD40 cells as prepared in Example 1 were seeded into eachwell of the 96-well plates and hybridoma cell culture supernatantdiluted 10 times with dilution buffer (PBS plus 1% BSA, 1% goat serum,and 0.01% Tween 20) was added to the plates (100 μl/well). Afterincubated at 4° C. for 1 hour, plates were washed 3 times with PBST.Then, cells were added with an APC goat anti-mouse IgG (BioLegen, US,Cat #: 405308) diluted 500× was added to the plates. After incubation at4° C. for 1 hour, plates were washed with PBS for 3 times and then thecell fluorescence was monitored using a FACS machine (BD).

Based on the FACS screening, 162 positive clones were obtained thatdisplayed high binding capacity to both HEK293A/humanCD40 andHEK293A/rhesusCD40 cells.

Subcloning of Hybridoma Clones Producing Anti-CD40 Antibodies

The 162 hybridoma clones were subject to 2 rounds of subcloning. Duringthe subcloning, multiple subclones (n>3) from each parent clone wereselected and confirmed by ELISA and FACS assays as described above. Thesubclones selected through this process were defined as hybridoma cellsproducing monoclonal antibodies. Finally, 108 subclones (one subclonefrom each parent clone) having high binding capacity to both human andmonkey CD40 were obtained.

Screening of Hybridoma Cell Lines by HEK Blue Activity Assay

The 108 subclones were expanded in 96-well plates and then cultured for5 days. Supernatants were harvested for HEK-Blue activity assays toidentify CD40 antibodies having agonist activity to human CD40.

Briefly, a stable HEK-Blue reporter cell line expressing human CD40 (SEQID NO.: 68) (referred to as HEK-Blue/CD40) was established by infectingHEK-Blue null 1_v cells (InvivoGen, San Diego, Calif.) withCD40-expressing lentivirus (which was generated in Example 1), followedby selection with 10 μg/ml puromycin.

For HEK-Blue reporter assay, 40000 HEK-Blue/CD40 cells resuspended in200 μl of culture media (DMEM medium (Hyclone, USA, Cat #:SH30243.01)+10% FBS (Excell, China, Cat #: FND500)+10 μg/ml Puromycin(GIBCO, USA, Cat #: A11138-03)+100 μg/ml Normocin™ Invivogen, USA, Cat#: ant-nr-2)+100 μg/ml Zeocin (Invivogen, USA, Cat #: ant-Zn-5)) wereplated in a 96-well plate and cultured at 37° C. overnight. On the2^(nd) day, 200 μl of DMEM medium was added to each well to replace theculture medium. Seven hours later, the DMEM medium in the well wasreplaced with 100 μL/well of HEK Blue Detection buffer (Invitrogen; US;Cat #: hb-det3) and 100 μL/well of hybridoma cell culture supurnatant.The resultant mixtures were incubated at 37° C. under 5% CO₂ until theappropriate blue color developed. OD630 was measured using a SpectraMaxmicroplate reader (Molecular Devices; US; SpectraMaxR i3X). An anti-HELantidbody (LifeTein, LLC, US, Cat. #:LT12031) was used as negativecontrol, and RO7009789 (an agonistic antibody, prepared using amino acidsequences disclosed in U.S. Pat. No. 7,338,660B2 with human IgG2/kappaconstant regions) and CD40L (Sino Biological, China, Cat:10239-H08E),the natural ligand and activator of CD40, were used as positivecontrols. As shown in FIG. 1, 38 clones displayed different levels ofCD40 agonist activity while others showed no agonistic activity.

Example 3 Purification of Mouse Anti-CD40 Monoclonal Antibodies

Based on the HEK-Blue assays as mentioned above, 20 clones (see Table 3below) with high HEK-Blue activity were selected for furthercharacterizations. Monoclonal mouse antibodies from the 20 selectedclones were purified. Briefly, hybridoma cells of each subclone weregrown in T175 cell culture flasks each having 100 ml of fresh serum-freemedium (Gibco, US, Cat #: 12045-076) with 1% HT supplement (Gibco, Cat#: 11067-030). Cell cultures were kept for 10 days in an incubator with5% CO₂ at 37° C. Cell cultures were collected, followed bycentrifugation at 3500 rpm for 5 minutes and then subject to filtrationusing a 0.22 μm capsule to remove the cell debris. Monoclonal mouseantibodies were then purified using a pre-equilibrated Protein-Aaffinity column (GE, USA, Cat #: 17040501) and eluted with elutionbuffer (20 mM citric acid, pH3.0-pH3.5). Then, antibodies were kept inPBS buffer (pH 7.0), and their concentrations were determined using aNanoDrop instrument.

The isotype of each purified antibody was determined by using the RapidIsotyping Kit with Kappa and Lambda-Mouse (Thermal, USA, Cat #: 26179)and Mouse Monoclonal Antibody Isotyping Reagents (Sigma, USA, Cat #:IS02-1KT), following the manufacturer's manuals. The isotyping resultsand the expression titer of the selected top 20 clones were summarizedin Table 3.

TABLE 3 Isotype and expression titer of anti-OD40 antibodies Expressiontiter clone Isotype (mg/L) 13A2 mouse IgG1/κ 24.744 16A6 mouse IgG1/κ31.111 29A10 mouse IgG1/κ 33.889 7B4 mouse IgG1/κ 18.667 9A7 mouseIgG1/κ 7.778 19H4 mouse IgG1/κ 10.000 37G10 mouse IgG1/κ 65.333 35C9mouse IgG1/κ 11.667 16F4 mouse IgG2b/κ 14.000 50F6 mouse IgG1/κ 12.77877D9 mouse IgG1/κ 12.22 79D7 mouse IgG1/κ 20.39 142F7 mouse IgG1/κ 17.2289D11 mouse IgG1/κ 95.73 91E4 mouse IgG2a/κ 4.47 101C12 mouse IgG1/κ18.94 92F6 mouse IgG1/κ 39.97 82D3 mouse IgG2a/κ 16.27 23B8 mouseIgG2a/κ 32.33 51F7 mouse IgG1/κ 1.44

Example 4 Purified Mouse Anti-CD40 Monoclonal Antibodies Bound to Humanand Monkey CD40

Purified mouse anti-CD40 monoclonal antibodies were firstlycharacterized by ELISA assays to determine their binding affinities torecombinant human, monkey or mouse CD40 proteins.

ELISA plates were coated with 500 ng/ml human CD40 (ECD)-his (SinoBiological, CN, Cat #: 10774-H08H) at 4° C. overnight. The wells wereblocked with 200 μl of blocking buffer (PBS containing 1% BSA, 1% goatserum, and 0.05% Tween 20) for 2 hours at room temperature, and then 100μl of serially diluted anti-CD40 antibodies (starting from 40000 ng/ml)were added to each well and incubated for 1 hour at RT. Plates werewashed for 3 times with PBST (PBS+0.05% Tween 20), added withGoat-anti-mouse IgG-HRP (Simga, US, Cat #: A9309-1 ml) diluted 5000×,and incubated for 1 hour at RT. Plates were developed with freshlyprepared Ultra-TMB (BD, US, Cat #:555214) for 5 minutes at RT.Absorbance was read on a SpectraMax® i3X (Molecular Devies, US) at 450nm.

Species-cross-reactivity of the 20 CD40 mAbs to monkey or mouse CD40 wasfurther assessed by direct ELISA. Briefly, 500 ng/ml monkey CD40(ECD)-hFc (Sino Biological, CN, Cat #: 90097-C02H) or mouse CD40-hFc(Sino Biological, CN, Cat #: 50324-M03H) was coated on 96-well ELISAplates followed by incubation with 100 μl of serially diluted anti-CD40antibodies (starting from 40000 ng/ml). Goat anti-mouse IgG conjugatedwith HRP (Sigma, US, Cat #:A9309-lml) was used then. Anti-CD40antibodies RO7009789 and ADC1013 (prepared using the amino acidsequences disclosed in US2016/0311916A1 with human IgG1/kappa constantregions) were used as references.

EC₅₀ values for these binding tests were summarized in Table 4. It canbe seen that all the 20 antibodies, except 51F7, clearly cross-reactedwith monkey CD40 but not with mouse CD40.

TABLE 4 Binding capacity of 20 mouse anti-CD40 mAbs to human, monkey ormouse CD40 ELISA (EC₅₀:ng/ml) hCD40 rhCD40 muCD40- Clone (ECD)-his(ECd)-hFc hFc RO7009789 20.65 16.16 N/A ADC1013 24.03 22.92 N/A 9A717.39 1349 N/A 16A6 13.12 12.95 N/A 19H4 26.09 20.46 N/A 29A10 31.8230.21 N/A 16F4 25.68 23.38 N/A 35C9 21.64 21.33 N/A 50F6 15.52 16.02 N/A7B4 15.29 15.25 N/A 13A2 20.65 14.08 N/A 37G3 24.03 30.15 N/A 77D9 21.3720.56 N/A 79D7 15.64 17.02 N/A 142F7 17.12 20.14 N/A 89D11 156.7 142.7N/A 91E4 17.68 18.87 N/A 101C12 19.09 19.77 N/A 92F6 29.11 36.55 N/A82D3 15.26 17.64 N/A 23B8 17.76 18.42 N/A 51F7 47.87 N/A N/A

Example 5 Mouse Anti-CD40 Monoclonal Antibodies Bound to Human andRhesus CD40 Expressed on HEK293A Cells

To further determine whether anti-CD40 antibodies bound to human, monkeyor mouse CD40 expressed on HEK293A cells, a cell-based binding assay byFACS was performed using the HEK293A cells stably overexpressing human,monkey or mouse CD40 as generated in Example 1, respectively. Briefly,10⁵ HEK293A cells were seeded into each well of the 96-well plates andserially diluted anti-CD40 antibodies were added to the plates. Afterincubated at 4° C. for 1 hour, plates were washed 3 times with PBST.Then, an APC coupled Goat Anti-Mouse IgG (BioLegen, US, Cat #:405308)diluted 500× was added to the plates. After incubation at 4° C. for 1hour, the plates were washed with PBS for 3 times and then cellfluorescence was monitored using a FACS machine (BD).

As shown in Table 5 below, all of the mouse anti-CD40 monoclonalantibodies showed high binding capacity to both human and rhesus monkeyCD40 but did not bind to mouse CD40 (data not shown).

TABLE 5 Binding affinity of mouse anti OX-40 antibodies to human andmonkey CD40 FACS(EC₅₀: ng/ml) HEK-293A/h HEK-293A/Rh Clone CD40 CD40R07009789 227.4 184.8 ADC1013 148.7 67.29 9A7 90.53 6876 16A6 68.2144.03 19H4 227.7 206.6 29A10 202.6 184.5 16F4 199.5 125.7 35C9 181.8 17250F6 134.1 136.6 7B4 10.09 15.87 13A2 48.45 84.27 37G3 203.2 185.6 77D933.03 117.2 79D7 12.35 78.42 142F7 15.46 80.66 89D11 58.03 83.58 91E448.66 101.8 101C12 21.87 62.63 92F6 77.86 129.6 82D3 21.77 130.7 23B81029 1034 51F7 108.6 483.8

Example 6 Mouse Anti-CD40 Antibodies Inhibited or Promoted HumanCD40-CD40L Interaction

Purified anti-CD40 antibodies were further analyzed for their ability ofblocking or promoting binding of human CD40L to human CD40. Briefly,96-well ELISA plates were coated with 500 ng/ml human CD40L (SinoBiological, China, Cat:10239-H08E) at 4° C. overnight. The plates wereblocked with 200 μl of blocking buffer (PBS+2% BSA) for 2 hours at roomtemperature. Then serially diluted anti-CD40 antibodies (sarting from 40μg/ml) were mixed and incubated with 2 μg/ml human CD40-hFc (Sinobiological, Cat #:10774-H02H) at 37° C. for 1 hour, which mixtures werethen added into the wells and incubated at RT for 1 hour. The plateswere washed 3 times with PBST (PBS+0.05% Tween20), added with anti-HumanIgG FC-HRP (1:5000, Sigma, USA, Cat #: A0170-1 ML), and then incubatedat room temperature for 1 hour. Plates were washed 3 times with PBST andthen 80 μl of TMB was added to each well. Five to ten min later, 80 μlof 0.16 M sulfuric acid was added to each well and then OD450 wasmeasured on a SpectraMaxR i3X (Molecular Devies, US).

Interestingly, the data showed that 6 mouse antibodies (13A2, 16A6, 7B4,50F6, 142F7 and 101C12) promoted the human CD40/CD40L interactions whileanother 3 antibodies (23B8, 92F6, 82D3) blocked the CD40-CD40Linteractions, with the remainings having no evident influence onCD40-CD40L interaction. Results of 4 representative antibodies wereshown in FIGS. 2A and 2B.

Example 7 Determination of Agonistic Activity of Mouse Anti-CD40Antibodies

To determine whether the selected mouse anti-CD40 antibodies hadagonistic activity, a HEK-Blue activity assay was performed. Briefly,the HEK-Blue/CD40 cells, generated in Example 2, were incubated in DMEMmedium (Hyclone, USA, Cat #: SH30243.01)+10% FBS (Excell, China, Cat #:FND500)+10 μg/ml Puromycin (GIBCO, USA, Cat #: A11138-03)+100 μg/mlNormocin™ (Invivogen, USA, Cat #: ant-nr-2)+100 μg/ml Zeocin (Invivogen,USA, Cat #: ant-Zn-5). Forty-thousand (40,000) HEK-Blue/CD40 cells in200 μl of culture medium were aliquoted in each well of the 96-wellassay plate and cultured at 37° C. After overnight incubation (˜12hour), 200 μl of fresh DMEM medium was used to replace the culturemedium. Seven hours later, the DMEM medium in each well was replacedwith 100 μL/well of HEK Blue Detection buffer (Invivogen; USA; Cat #:hb-det3) containing anti-CD40 antibodies at various concentration (from100 μg/ml to 0.01 ng/ml). The cells were incubated at 37° C. untilappropriate blue color developed. Absorbence at 630 nm was measuredusing a SpectraMax microplate reader (Molecular Devices, US, SpectraMaxRi3X).

EC₅₀ values were summarized in Table 6 below, and representative curveswere shown in FIG. 3. As can be seen, all 20 mouse antibodies displayeddifferent agonist activities in the HEK-Blue assay, suggesting theirabilities in simulating CD40 downstream signalings.

TABLE 6 Agonistic activity of anti-CD40 antibodies Antibody HEK BlueEC₅₀(ng/mL) RO7009789 23.26 ADC1013 1034 13A2 12.61 16A6 11.37 7B4 26.1450F6 42.16 101C12 175.2 77D9 22.26 35C9 86.08 37G3 146.1 79D7 55.29142F7 64.77 89D11 91.06 91E4 152.7 82D3 285.4 23B8 3373 51F7 6173 92F6263.7 19H4 92.79 16F4 252.1 29A10 139.8 9A7 61.96

Example 8 Epitope Binning

For epitope binning, a competition ELISA assay was performed. Briefly,96-well plates were coated with 5 μg/ml RO7009789 or ADC1013 at 4° C.overnight. The wells were blocked with 200 μl of blocking buffer (PBScontaining 1% BSA, 1% goat serum, and 0.05% Tween 20) for 2 hours atroom temperature. Human CD40 (ECD)-His (Sino Biological, CN, Cat#:10774-H08H) was diluted to 0.5 μg/mL and added to the plate which wasthen incubated for 1 hour at RT. The ELISA plates were washed for 3times with PBST, and then the purified antibodies were diluted to 1μg/mL and added to each well and allowed to incubate for 1 hour at RT.The ELISA plates were washed for 3 times with PBST, and then anti-mouseFc-HRP (Sigma, US, Cat #: A9309-1MC) diluted at 1:20000 was added toeach well and incubated for 1 hour at RT. Plates were developed withfreshly prepared Ultra-TMB (Huzhou Yingchuang, CN, Cat #: TMB-S-003) for5 minutes at RT and the absorbance was measured on SpectraMax microplatereader (Molecular Devices; US; SpectraMaxR i3X) at 450 nm (OD450).

The results were summarized in Table 7. Seven mouse antibodies (101C12,142F7, 89D11, 13A2, 16A6, 7B4 and 50F6) competed with both referenceantibodies while antibodies 9A7, 92F6, 19H4, 16F4 and 51F7 did not showcompetition with either reference antibody. The remaining antibodiescompeted with either of the two reference antibodies.

TABLE 7 Epitope binning by competition ELISA Binning results AntibodiesR07009789 ADC1013 13A2 + + 16A6 + + 7B4 + + 50F6 + + 101C12 + + 77D9 + −35C9 + − 37G3 + − 29A10 + − 9A7 − − 92F6 − − 19H4 − − 16F4 − − 79D7 − −142F7 + + 89D11 + + 91E4 + − 82D3 + − 23B8 + − 51F7 − − +: withcompetition; −: without competition

Example 9 Agonistic Anti-CD40 Antibodies Drove Dendritic Cell Maturation

To further determine the agonistic activity of the mouse anti-CD40antibodies, a dendritic cell maturation assay was performed. Briefly,PBMCs from one healthy human donor's blood sample were collected bydensity gradient centrifugation and then resuspended in RPMI1640 medium.PBMCs were cultured n a 37° C. incubator for 2 hours, and cells adheredto container walls were collected as isolated monocytes. The monocyteswere cultured with 100 ng/ml of recombinant human GM-CSF (R&D, US, Cat#: 7954-GM) and 100 ng/ml of recombinant human IL-4 (R&D, US, Cat #:6507-IL) in RPMI1640 media supplemented with 10% FBS in a 24-well plate.Three days later, half of the medium was replaced with fresh medium. Onday 6 of culturing, anti-CD40 antibodies (10 μg/ml or 1 μg/ml), or thecontrol antibodies (RO7009789, ADC1013 and Hel (LifeTein, US, Cat #:LT12031)) were added to the cells, and the plate was further culturedfor 48 h. FITC Mouse Anti-human CD83 (BD, USA, Cat #: 556910), PE MouseAnti-human CD86 (BD, Cat #: 555658), and BV650 Mouse Anti-human CD80(BD, USA, Cat #: 564158) were used for staining of DC activation markersby FACS.

Results of representative antibodies were shown in FIG. 4A-4C. Mouseanti-CD40 antibodies 16A6, 29A10, 7B4 and 13A2 increased the expressionof CD86, a biomarker of maturated dendritic cells, as compared toanti-Hel isotype control, and antibodies 16A6, 29A10, 7B4 and 13A2significantly up-regulated expression of CD80 and CD83, both beingco-stimulatory molecules.

Example 10 Expression and Purification of Chimeric Anti-CD40 Antibodies

Eight antibodies (13A2, 16A6, 7B4, 29A10, 92F6, 77D9, 50F6 and 142F7)were selected for further tests. The variable region sequences of the 8selected candidate antibodies were cloned from hybridoma cells using thestandard PCR method with a set of degenerated primers as describes inliteratures (Juste et al., (2006), Anal Biochem. 1; 349(1):159-61).Expression vectors were constructed by inserting the sequences encodingthe variable region sequences plus respective human IgG2/kappa constantregion sequences (amino acid sequences of heavy chain constant regionand light chain constant region set forth in SEQ ID NOs: 63 and 65,respectively) into XhoI/BamHI restriction sites of pCDNA3.1 (Invitrogen,Carlsbad, USA). The amino acid SEQ ID numbers of variable regions weresummarized in Table 1 above.

The expression vectors were PEI transfected into HEK-293F cells(Cobioer, NJ, China). In specific, HEK-293F cells were cultured in FreeStyle™ 293 Expression Medium (Gibco, Cat #: 12338-018) and transfectedwith the expression vectors using polyethyleneinimine (PEI) at a DNA:PEIratio of 1:3, 1.5 μg of DNAs per millimeter of cell medium. TransfectedHEK-293F cells were cultured in an incubator at 37° C. under 5% CO₂ withshaking at 120 RPM. After 10-12 days, supernatants were harvested andmonoclonal antibodies were purified as described in Example 3.

Example 11 Chimeric Anti-CD40 Monoclonal Antibodies Bound to Human orRhesus Monkey CD40 Expressed on HEK293A Cells

The chimeric anti-CD40 antibodies were further characterized for theirability of binding to HEK293A/humanCD40 cells, HEK293A/rhesusCD40 cellsand HEK293A/mouseCD40 cells as generated in Example 1, according to theprotocol of Example 5.

As shown in FIGS. 5A and 5B, the chimeric antibodies had high bindingaffinity to both human and monkey CD40. These chimeric antibodies didnot bind to mouse CD40 (data not shown).

Example 12 Chimeric Anti-CD40 Monoclonal Antibodies had AngonistActivity and Drove Dendritic Cell Maturation

The chimeric antibodies were assayed for their effect on CD40 signalingactivation by HEK-Blue assay and dendritic cell maturation assay,following the protocols described in Example 7 and Example 9. RO7009789,APX005 (prepared using the amino acid sequences disclosed inWO2014/070934A1 having human IgG1/kappa constant regions) and/or ADC1013were used as references.

As shown in FIG. 6, the 8 chimeric antibodies displayed similarfunctional activities as their parent monoclonal antibodies. FIG. 7showed that all tested chimeric antibodies drove maturation of dendriticcells, as suggested by upregulation of CD86, the biomark of maturateddendritic cells.

Example 13 Humanization of Anti-CD40 Antibodies

Based on the characterizations and assays described above, two candidateantibodies, 7B4 and 13A2, were selected for humanization and furtherinvestigations. Humanization of the murine antibodies was conductedusing the well-established CDR-grafting method (U.S. Pat. No. 5,225,539,incorporated herein by reference in its entirety) as described in detailbelow.

To select acceptor frameworks for humanization of murine antibodies 7B4and 13A2, the light and heavy chain variable region sequences of 7B4 and13A2 were blasted against the human immunoglobulin gene database in NCBIwebsite (http://www.ncbi.nlm.nih.gov/igblast/). The human germline IGVHand IGVK with the highest homology to 7B4 and 13A2 were selected as theacceptor for humanization. For antibodies 7B4 and 13A2, the human heavychain acceptor selected was IGHV4-28*06, and the human light chainacceptor selected was IGKV2-30*02 listed in Table 8 below.

The three dimensional structures were simulated for variable domains of7B4 and 13A2 in order to identify key framework residues that might beplaying important roles in supporting CDR loop structures, thusdesigning back mutations in humanized antibodies. Selected structuretemplates had the same classes of canonical loop structures in L-CDR1,L-CDR2, L-CDR3, H-CDR1, H-CDR2 and H-CDR3 to 7B4 and 13A2, respectively.Using the structural templates selected, structural models were built byreplacing the murine frameworks with human acceptor's frameworks forheavy and light chains. Three-dimensional structural modeling simulationwas then performed to identify key framework residues that might beimportant in supporting the CDR-loop structures or the heavy and lightchain interface. When the murine antibody and the human acceptor sharedthe same residue at a certain site in the framework, the human germlineresidue was kept. On the other hand, when the murine antibody and humangermline acceptor had a different residue at a certain site in theframework, the importance of this residue was evaluated by structuralmodeling. If a residue in the murine antibody's framework was found tointeract with and influence the CDR residues, then this residue wasback-mutated to murine residue.

TABLE 8 Structural templates used in antibody structure simulations PDBcode of Sequence Sequence Antibody chain template structure identitysimilarity 13A2 Heavy chain 5E2T 71% 83% 13A2 Light chain 1DLF 84% 92%7B4 Heavy chain 5E2T 87% 90% 7B4 Light chain 1DLF 87% 95%

Based on the structural modeling as described above, 5 potentialback-mutations (I49M, V68I, M70I, K44N, G45K) were identified for heavychain and 5 potential back-mutations (M4L, R51L, F76L, Y92F, Q105S) forlight chain of 13A2. For 7B4, 5 potential back-mutations (I49M, V68I,M70I, K44N, G45K) were identified for heavy chain and 4 back-mutations(M4L, R51L, Y92F, Q105S) were identified for light chain.

As summarized in Table 1, for both 7B4 and 13A2, three humanized heavychain variable regions and three humanized light chain variable regionswere designed, with a total of 5 humanized antibodies.

The sequences encoding the humanized heavy chain variable region plushuman IgG2 constant region, and the sequence encoding light chainvariable regions plus human kappa constant region (amino acid sequencesof heavy chain constant region and light chain constant region set forthin SEQ ID NOs: 63 and 65, respectively) were chemically synthesized andthen subcloned into the pcDNA3.1(+)-based expression vector (Invitrogen,USA) using the BamH I and Xho I restriction sites, respectively. Allexpression constructs were confirmed by DNA sequencing. The HEK293Fexpression systems (Invitrogen, USA) were transfected with heavy chainand light chain expressing vectors and transiently expressed 10humanized anti-CD40 antibodies (5 for 13A2, and 5 for 7B4), according tothe protocol described in Example 10. The humanized antibodies werepurified as described in Example 3.

Example 14 Characterization of Chimeric and Humanized Anti-CD40Antibodies

The chimeric and humanized anti-CD40 antibodies were furthercharacterized for their ability of binding to HEK293A/humanCD40 cellsand HEK293A/rhesus CD40 cells, following the protocols described inExample 5. They were also tested for their ability to activate CD40signaling in HEK-Blue assay, and to promote dendritic cell maturation,following the protocols in Example 7 and Example 9, respectively, thedendritic cells collected form three healthy human donors. IL-12(p40)secretion by the dendritic cells was measured by using human IL12(p40)ELISA kit (BD, US, Cat #:551116) following themanufactor's instruction.

As shown in FIG. 8A-8D, FIG. 9A-9B, FIG. 10A-10C (Donor 1), FIG. 11A-11B(Donor 2), and FIG. 12A-12C (Donor 3), the humanized anti-CD40antibodies 13A2-VH3VL2, 13A2-VH3VL3, and 7B4H2L2 showed best binding,agonistic and functional activities.

Example 15 Affinity of Chimeric or Humanized Anti-CD40 Antibodies toHuman CD40

SPR assays were used to determine the binding affinity of the chimeticor humanized anti-CD40 antibodies to human CD40 with the BIAcore™ 8Kinstrument (GE Life Sciences). Briefly, 100-200 response units (RU) ofhuman CD40 (ECD)-his protein (Sino Biological, CN, Cat #:10774-H08H)were coupled to CMS biosensor chips (Cat #: BR-1005-30, GE LifeSciences), followed by blocking of un-reacted groups with 1Methanolamine. Serially diluted antibodies at concentrations ranging from0.3 μM to 10 μM were injected into the SPR running buffer (HBS-EPbuffer, pH7.4, GE Life Sciences; US; Cat #:BR-1006-69) at 30 μL/minute.The binding capacitity was calculated with the RUs of blank controlssubtracted. The association rate (k_(a)) and dissociation rate (k_(d))were calculated using the one-to-one Langmuir binding model (BIAEvaluation Software, GE Life Sciences). The equilibrium dissociationconstant K_(D) was calculated as the k_(d)/k_(a) ratio. TheSPR-determined binding curves of antibodies were shown in FIGS. 13A-13N,and the binding affinities of those chimeric or humanized antibodieswere listed in Table 9.

TABLE 9 Binding affinities of anti-CD40 antibodies to human CD40Antibodies k_(a) k_(d) K_(D) RO700789 1.07E+5 1.83E−4 1.71E−9 ADC10131.2E+6 3.48E−2 2.9E−8 13A2 4.84E+05 1.73E−03 3.58E−09 13A2-VH0VL03.96E+05 1.14E−02 2.88E−08 13A2-VH2VL2 8.23E+05 1.71E−03 2.08E−0913A2-VH2VL3 7.35E+05 2.61E−03 3.55E−09 13A2-VH3VL2 8.25E+5 2.42E−32.93E−9 13A2-VH3VL3 5.98E+5 4.59E−3 7.68E−9 7B4 1.00E+06 3.6E−03 3.6E−097B4-VH0VL0 2.75E+06 6.8E−03 2.47E−09 7B4-VH2VL2 2E+06 5.81E−03 2.91E−097B4-VH2VL3 1.78E+06 6.39E−03 3.6E−09 7B4-VH3VL2 1.46E+06 6.15E−034.2E−09 7B4-VH3VL3 2.13E+06 4.88E−03 2.99E−09

Example 16 Epitope Mapping of Chimeric or Humanized Anti-CD40 Antibodies

The chimeric and humanized anti-CD40 antibodies were tested for theirbinding epitope by ELISA.

There are four individual cysteine enriched domains (CRD) in CD40extracellular domain (ECD), namely CRD1, CRD2, CRD3 and CRD4. Based onthe structure of CD40 ECD, one full-length CD40 ECD, five CD40 ECDtruncants and four CD40 ECD mutants were generated, and theirinformation can be found in Table 10 below. These recombinant proteinswere linked with a signal peptide (SEQ ID NO.: 83) at the N terminus forprotein secretion and a mFc-tag (SEQ ID NO.: 84) at the C terminus forELISA assay. DNA sequences encoding these recombinant proteins weresynthesized and subcloned into pcDNA3.1 vector. The expression andpurification of the recombinant proteins were carried out according tothe protocols in Example 10. ELISA assay was performed to assess bindingcapacity of mAbs to recombinant CD40 proteins, following the protocol inExample 2.

As shown in FIG. 14A, all the antibodies bound to full-length CD40 ECD,but none of them bound to the truncants, indicating CRD1 domain of CD40was involved in and important to the binding of antibodies. FIG. 14Bshowed that the chimeric 13A2 antibody, the three humanized antibodiesand ADC1013 cannot bind to CD40 Mutant 2-4, indicating the fiveantibodies shared the same or similar binding epitope. RO7009789 cannotbind to Mutant 1, 2 and 4 while APX005 cannot bind to Mutant 2 and 4.

TABLE 10 CD40 ECD truncants and mutants SEQ CD40 ECD recombinant proteinID NO. CRD Mutation Full-length CD40 ECD 73 CRD1/2/3/4 n/a (amino acid21-193, human CD40) Truncant 1 (amino acid 61-193) 74 CRD2/3/4 n/aTruncant 2 (amino acid 104-193) 75 CRD3/4 n/a Truncant 3 (amino acid145-193) 76 CRD4 n/a Truncant 4 (amino acid 38-193) 77 CRDA1/2/3/4 n/aTruncant 5 (amino acid 21-37 & 78 CRDA1/2/3/4 n/a 61-193) Mutant 1(amino acid 21-193) 79 CRD1/2/3/4 R7A/E8A Mutant 2 (amino acid 21-193)80 CRD1/2/3/4 T32A/E33A Mutant 3 (amino acid 21-193) 81 CRD1/2/3/4F34A/T35A Mutant 4 (amino acid 21-193) 82 CRD1/2/3/4 E35A/T37A Note: CRDΔ1: truncated CDR1 domain

Example 17 Humanized Anti-CD40 Antibodies Specifically Bound to HumanCD40

ELISA assays were performed to determine the binding specificity ofanti-CD40 antibodies to human CD40, in comparison to other human TNFRSFmembers with homologous amino acid sequences, following the protocoldescribed in Example 2.

The binding affinities of anti-CD40 antibodies to human CD40 (ECD)-his(TNFRSF5, Sino Biological, China, Cat #:10774-H08H), human OX40-his(TNFRSF4, Sino Biological, China, Cat #:10481-H08H), human HVEM-mFc(TNFRSF14, ACRO, China, Cat #: HVM-H5255), human 4-1BB (TNFRSF9, ACRO,China, Cat #: 41B-H522a), human NGFR (TNFRSF16, Sino Biological, China,Cat #: 13184-H08H), human DR6 (TNFRSF21, Sino Biological, China, Cat #:10175-H08H), human RANK(TNFRSF11, ACRO, China, Cat #: RAL-H5240) werestudied.

As shown in FIGS. 15A and 15B, neither 13A2VH3VL3 nor 7B4VH2VL2 showedbinding to recombinant human OX40 (TNFRSF4), HVEM(TNFRSF14),4-1BB(TNFRSF9), NGFR(TNFRSF16), DR6(TNFRSF21) or RANK(TNFRSF11),suggesting that 13A2VH3VL3 and 7B4VH2VL2 specifically bound to humanCD40.

Example 18 Engineered Anti-CD40 Antibodies had Better Agonistic Activity

It has been suggested that optimal biological and anti-tumor effects ofagonistic anti-CD40 antibodies require Fc receptor (FcR) coengagement(Richman and Vonderheide, (2014) Cancer Immunol Res 2(1): 19-26). Thus,anti-CD40 antibodies were prepared having heavy/light chain variableregion of 13A2VH3VL3 or 7B4VH2VL2 plus human IgG1/kappa constant region,wherein the human IgG1 constant region was engineered to have S267E andL328F mutations (mutated IgG1 constant region's amino acid sequence setforth in SEQ ID NO.: 64). The obtained antibodies were named as13A2-VH3VL3-IgG1(SE/LF) and 7B4-VH2VL2-IgG1(SE/LF), respectively, andwere further characterized for their ability of promoting dendritic cellmaturation, following the protocal in Example 9. RO7009789, ADC1013 andAPX005 were used as positive controls, and Hel was used as negativecontrol.

As shown in FIGS. 16A, 16B and 16C, 13A2-VH3VL3-IgG1(SE/LF) and7B4-VH2VL2-IgG1(SE/LF) both showed significantly higher agonist activityin promoting dendritic cell maturation than their parents' antibodies,with 13A2-VH3VL3-IgG1(SE/LF) having the highest activity among alltested antibodies.

Example 19 Humanized Anti-CD40 Antibodies had In Vivo Anti-Tumor Effect

In vivo anti-tumor activity of anti-CD40 antibodies having heavy/lightchain variable region of 13A2-VH3VL3 or 7B4-VH2VL2 and mouse IgG1/kappaconstant regions (amino acid sequences of mouse IgG1/kappa constantregions set forth in SEQ ID NOs.: 62 and 66, respectively) were studiedin an animal model established by grafting MC38 murine colonadenocarcinoma in transgenic mice with human CD40 (GemPharmatech Co.Ltd, China). Mouse IgG1/kappa constant regions were used to enhance Fcfunction in the mouse model. Each mouse was subcutaneously injected with1×10⁶ MC38 cells at one flank at day 0. When tumors grew to about 80mm³, the animals were randomly assigned into five groups, 8 mice pergroup. The animals were then i.p. administered with 13A2-VH3VL3,7B4-VH2VL2, RO7009789, APX005 or PBS at a dose of 10 mg/kg/day at Day 4,7, 11, 14, 18 and 21, RO7009789 and APX005 both engineered to have mouseIgG1/kappa constant regions (amino acid sequences of mouse IgG1/kappaconstant regions set forth in SEQ ID NOs.: 62 and 66, respectively).

Tumor size and mice body weight were followed over time. Tumormeasurements (width and length) were taken by caliper and tumor volumecalculated by the formula TV=(length×width²)/2. The experiment wasterminated before the tumor volume in antibody administration groupsreached 3.5 cm³. One-way ANOVA was used to identify tumor volumedifferences.

At Day 25, four mice with bigger tumors from each group were selectedfor T cell analysis. The tumors were collected immediately after themice were sacrificed and placed in Hanks buffer with collagenases. Thetumors were then cut into small pieces and incubated in Hanks bufferwith collagenases at 37° C. for 30 min with gentle shaking. Thereafter,10 ml of RPMI 1640+10% FBS was added to each sample to deactivate thecollagenase and maintain viability of the immune cells. Samples werepassed through a 70 μm cell filter membrane (Corning, Cat #: 352350) andplaced in new tubes. The samples were pelleted and resuspended in PBSFbuffer (PBS+2% FBS) at a density of 1*10⁷ cells/ml. The samples werewashed by PBSF buffer for 2 times. Each sample was divided into twoparts, one added with anti-CD45 (Brilliant Violet 785 ™ anti-mouse CD45Antibody; Biolegend; US; Cat #: 103149), anti-CD8 (APC anti-mouse CD8aAntibody; Biolegend; US; Cat #: 100712), anti-CD3 (FITC anti-mouse CD3Antibody; Biolegend; US; Cat #: 100203) and anti-CD4 (PerCP anti-mouseCD4 Antibody; Biolegend; US; Cat #: 100432) fluorescent antibodymixtures, and another added with anti-CD11c (APC anti-mouse CD8aAntibody; Biolegend; US; Cat #: 100712), anti-CD80 (APC anti-mouse CD11cAntibody; Biolegend; US; Cat #: 117310), anti-CD83 (PE/Cy7 anti-mouseCD83 Antibody; Biolegend; US; Cat #: 121518) and anti-CD86 (FITCanti-mouse CD86 Antibody; Biolegend; US; Cat #: 105005) fluorescentantibody mixtures. The resultant mixtures were incubated for half anhour at 4° C. Cells were washed 2 times by PBSF buffer and analyzed on aFACS machine (BD).

As shown in FIG. 17A-17F, treatment with anti-CD40 antibodiessignificantly reduced or inhibited tumor growth, as compared to negativecontrol group, although individuals responded differently. Tumor growthinhibition was observed in all mice (in the group with 7B4VH2VL2 orAPX005 administration) or most mice (6 out of 8 in the group with13A2VH3VL3 administration, 7 out of 8 in RO7009789 group). At Day 28,tumor vanished in all remaining 4 mice in 7B4VH2VL2 administration groupwhile 2 out of 4 in APX005 administration group had no tumor at all.

Treatment with anti-CD40 antibodies may cause mice body weight reductiondue to antibody's toxicity. As shown in FIG. 18 and Table 11 below, thebody weights of mice in 13A2VH3VL3 group at Day 25 increased a bitcompared to their initial weights, even if the tumor weights were takeninto consideration (tumors at Day 25 were about 1000 mm³ in size and 1.2g in weight), indicating antibody 13A2VH3VL3's low toxicity. In otherwords, treatment with 13A2VH3VL3 did not cause significant body weightreduction as observed in the RO7009789 group, and had less effect onmice body weight than that in the 7B4VH2VL2 or APX005 group.

TABLE 11 Mice body weight (Mean ± SE (g), 8 mice per group) over time infive groups Day Group 4 6 8 11 14 18 21 25 13A2-VH3VL3 22.9 ± 0.5 20.6 ±0.5 22.5 ± 0.5 24.1 ± 0.5 24.7 ± 0.5 25.2 ± 0.5 24.7 ± 0.5 25.4 ± 0.87B4-VH2VL2 23.0 ± 0.3 21.2 ± 0.2 23.4 ± 0.2 23.8 ± 0.2 22.7 ± 0.5 24.4 ±0.2 22.2 ± 0.5 22.9 ± 0.5 RO7009789 23.9 ± 0.3 21.5 ± 0.4 22.9 ± 0.422.6 ± 0.3 22.2 ± 0.4 22.1 ± 0.5 21.8 ± 0.4 19.6 ± 0.5 APX005 22.6 ± 0.320.8 ± 0.3 22.9 ± 0.3 23.1 ± 0.4 23.4 ± 0.6 24.1 ± 0.4 21.5 ± 0.6 22.0 ±0.7 PBS 23.3 ± 0.4 23.2 ± 0.3 24.2 ± 0.4 23.7 ± 0.3 25.1 ± 0.4 26.5 ±0.4 27.8 ± 0.5 30.7 ± 0.6

FIGS. 19A and 19B showed that antibody 7B4VH2VL2 evidently elevatedpercentage of both CD45⁺ CD3⁺ CD8⁺ cells and CD45⁺ CD3⁺ CD4⁺ cell inCD45⁺ cells. The percentage of CD45⁺ CD3⁺ CD8⁺ cells was also increasedin 13A2VH3VL3 treated mice. In addition, FIG. 20 indicated 7B4VH2VL2treatment significantly increased CD80 and CD83 expression on tumorinfiltrating dendritic cells (CD45⁺ CD11c⁺ cells), indicating its strongagonist activity in promoting dendritic cells maturation.

Amino acid sequences of some antibodies' heavy/light chain variableregions were summarized below.

Description/ Sequence/SEQ ID NO. VH for mouse, and chimeric 29A10antibodies (CDR regions underlined and bold)QVKLEQSGGGLVKPGGSLKLSCAASGFTF SHYYMY WVRQTPEKRLEWVA TISDAGSYTYYSDSVK GRFTISRDNAKNNLYLQMSSLKSDDTAMYFCARTYYRGD GGYWFFDV WGAGTAVTVSS (SEQ ID NO:40) VL for mouse, and chimeric 29A10 antibodies (CDR regions underlinedand bold) DIVITQSTSSLAVSVGEKVTMSC ESSQSLLYSSNQKNYL AWYQQKPGQSPKLLIYWASTRES GVPDR FTASGSGTDFTLTISSVKAEDLAVYYC QQYYRSPLT FGAGTKLELK (SEQ IDNO: 54)

While the invention has been described above in connection with one ormore embodiments, it should be understood that the invention is notlimited to those embodiments, and the description is intended to coverall alternatives, modifications, and equivalents, as may be includedwithin the spirit and scope of the appended claims. All referenced citedherein are further incorporated by reference in their entirety.

We claim:
 1. An isolated monoclonal antibody, or an antigen-bindingportion thereof, binding to tumor necrosis factor receptor CD40,comprising a heavy chain variable region comprising a VH-CDR1 region, aVH-CDR2 region and a VH-CDR3 region, and a light chain variable regioncomprising a VL-CDR1 region, a VL-CDR2 region and a VL-CDR3 region,wherein the VH-CDR1 region, VH-CDR2 region, VH-CDR3 region and theVL-CDR1 region, VL-CDR2 region and VL-CDR3 region comprise amino acidsequences of SEQ ID NOs: 3, 10, 16, 22, 28 and 32, respectively.
 2. Theisolated monoclonal antibody, or the antigen-binding portion thereof, ofclaim 1, wherein the heavy chain variable region comprises an amino acidsequence set forth in SEQ ID NO:
 40. 3. The isolated monoclonalantibody, or the antigen-binding portion thereof, of claim 1, whereinthe light chain variable region comprises an amino acid sequence setforth in SEQ ID NO:
 54. 4. The isolated monoclonal antibody, or theantigen-binding portion thereof, of claim 1, wherein the heavy chainvariable region and the light chain variable region comprise amino acidsequences set forth in SEQ ID NOs: 40 and 54, respectively.
 5. Theisolated monoclonal antibody, or the antigen-binding portion thereof, ofclaim 1, comprising a heavy chain constant region having an amino acidsequence set forth in SEQ ID NOs: 62, 63 or 64, linked to the heavychain variable region, and a light chain constant region having an aminoacid sequence set forth in SEQ ID NOs: 65 or 66, linked to the lightchain variable region.
 6. The isolated monoclonal antibody, or theantigen-binding portion thereof, of claim 1, which (a) binds CD40, (b)binds monkey CD40; (c) does not bind to mouse CD40; (d) activates CD40signaling; (e) promotes DC cell maturation; and (f) promotes CD4+ and/orCD8+ T cell proliferation.
 7. The isolated monoclonal antibody, or theantigen-binding portion thereof, of claim 1, which is a mouse, orchimeric antibody.
 8. The isolated monoclonal antibody, or theantigen-binding portion thereof, of claim 1, which is an IgG1, IgG2 orIgG4 isotype.
 9. A pharmaceutical composition comprising the isolatedmonoclonal antibody, or the antigen-binding portion thereof, of claim 1,and a pharmaceutically acceptable carrier.
 10. A method for activatingor inducing CD40 signaling to promote immune cell activation orproliferation in a subject having cancer, comprising administering tothe subject a therapeutically effective amount of the pharmaceuticalcomposition of claim
 9. 11. The method of claim 10, wherein the canceris a solid or non-solid tumor.
 12. The method of claim 10, wherein thecancer is selected from the group consisting of B cell lymphoma, chroniclymphocytic leukemia, multiple myeloma, melanoma, colon adenocarcinoma,pancreas cancer, colon cancer, gastric intestine cancer, prostatecancer, bladder cancer, kidney cancer, ovary cancer, cervix cancer,breast cancer, lung cancer, and nasopharynx cancer.
 13. The method ofclaim 10, further comprising administering an immunostimulatoryantibody, a costimulatory antibody, a chemotherapeutic agent, and/or acytokine.
 14. The method of claim 13, wherein the immunostimulatoryantibody is selected from the group consisting of an anti-VISTAantibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-LAG-3antibody, an anti-TIM 3 antibody, an anti-STAT3 antibody, and ananti-ROR1 antibody.
 15. The method of claim 13, wherein thecostimulatory antibody is an anti-CD137 antibody or an anti-GITRantibody.
 16. The method of claim 13, wherein the chemotherapeutic agentis epitubicin, oxaliplatin, and/or 5-fluorouracil.
 17. The method ofclaim 13, wherein the cytokine is GM-CSF and/or IL-4.